Cell senescence markers as diagnostic and therapeutic targets

ABSTRACT

Provided are methods and agents for depleting senescent cells endogenous to a subject, involving administering to the subject a binding agent that is selectively toxic to senescent cells in an amount effective to reduce the number of such cells, wherein the binding agent binds selectively to a senescent cell surface protein having a misfolded conformation, relative to said protein in a native conformation.

RELATED APPLICATIONS

This is a Patent Cooperation Treaty Application which claims the benefitof priority of U.S. Provisional Patent Application No. 61/827,255, filedMay 24, 2013 which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to markers of cell senescence, and toapplications thereof as targets for medical diagnosis and therapeuticintervention. More particularly, the invention relates to surfaceproteins that manifest in senescent cells, and to agents such asantibodies that bind such surface proteins for use in diagnosing andtreating related disease conditions.

BACKGROUND TO THE INVENTION

Senescence is generally defined as a cessation of cell replication thatfollows after a finite number of population doublings. Senescent cellsremain metabolically active but do not divide and resist apoptosis forlong periods. Cellular senescence is characterized by growth cyclearrest in the G1 phase, absence of S phase and lifespan control bymultiple dominant genes.

Cellular senescence differs from quiescence and terminaldifferentiation. Senescent cells manifest morphological changes such asenlargement, flattening and increased granularity. They do not divideeven if stimulated by mitogens. Replicative senescence is related to theprogressive shortening of telomeres. Senescence is induced when certainchromosomal telomeres reach a critical length, and can be abrogated bythe expression of telomerase which lengthens telomeres. Humanfibroblasts, for instance, undergo replication indefinitely whentransfected to express telomerase. Evidence suggests some relationshipbetween replicative senescence and aging.

There are pathways to senescence other than replication. These are oftenreferred to as stress-induced premature senescence (SIPS). Oxidativestress can shorten telomeres, and hyperoxia has been shown to inducesenescence. Gamma irradiation of human fibroblasts in early to mid G1phase causes senescence in a p53-dependent manner, and UV radiation alsoinduces cellular senescence. Other agents that can induce cellularsenescence include hydrogen peroxide, sodium butyrate, 5-azacytidine,and transfection with the Ras oncogene. Senescence in cancer cells canbe induced by such chemotherapeutics as doxorubicin, cisplatin, and5-bromodeoxyuridine, the latter being an agent that induces senescencein both normal and cancer cells.

The existence of cellular senescence has been demonstrated in vivo.Senescent fibroblasts were shown to exhibit staining for β-galactosidaseactivity at pH 6. These cells failed to incorporate tritiated thymidineand retained B-galactosidase activity after replating but did notdivide. Quiescent fibroblasts did not show staining. Keratinocytes,umbilical vein endothelial cells and mammary epithelial cells all showedincreased staining with increased population doublings. Immortalizedcells and terminally differentiated keratinocytes did not show staining.Staining was performed on skin biopsies to test whether senescence isobserved in vivo. An age-dependent pattern emerged in which an increasednumber of cells showed staining with increase donor age, in the dermisand epidermis. The existence of an increase in the number of senescentfibroblasts has been shown in the lungs of subjects with emphysema.

Cancer stem cells are immortal, meaning that they can replicateindefinitely without exhibiting senescence. Likely, the teleologicalpurpose of senescence is to prevent cancer by limiting the number ofcell divisions that can occur. Senescent cells can also promotetumorigenesis. Senescent stromal cells express tumour promoting as wellas tumour suppressing factors that exert an effect on neighbouringepithelial cells, such as mitogenicity and anti-apoptosis.

In a dramatic demonstration, Baker et al report that a variety ofage-related conditions were significantly improved in mice whensenescent cells were removed (Nature, 2011, 479:232-236). Treated micehad substantially delayed lordokyphosis (a measure of sarcopenia) andcataracts, as well as increased muscle fiber diameter, reduced loss ofadipose tissue, and improved skin tone. Some improvements were seen notonly in younger mice, but also in older recipients in which age-relatedconditions were already established.

In summary, cellular senescence does occur in vivo and is a likelysequel to environmental insults. Its prevalence increases with age atleast in some tissue compartments. Senescence confers functional changeson the cell which have been associated with various age-related diseasesthat include degenerative disorders such as Alzheimer's disease,cardiovascular disease, emphysema, sarcopenia, and tumorigenesis as wellas conditions more cosmetic in nature such as signs of skin agingincluding wrinkling, sagging, discolouration, and the like. Senescentcells also develop a so-called senescence associated secretory phenotype(SASP) which enables cells to secrete a variety of growth factors,cytokines, and proteases that contribute to age-related tissuedysfunction as well as to tumour formation, and thus create a localenvironment that is toxic to neighbouring normal cells.

There is accordingly a need for agents that are capable of detectingsenescent cells and for depleting senescent cells, thereby to diagnoseand treat conditions associated with cell senescence.

SUMMARY OF THE INVENTION

It has now been discovered that senescent cells manifest a surfacemarker that can be targeted for therapeutic and diagnostic purposes. Themarker is related to a surface protein that is native to the cell in itshealthy state. Particularly, it has been found that certain nativesurface proteins adopt a misfolded conformation when the host cell issenescent. Accordingly, the present invention exploits, as target, amisfolded form of a senescent cell surface protein. The misfoldedsurface protein is unique in conformational terms relative to the nativeprotein, and allows for the production and use of binding agents, suchas antibodies, that bind selectively to the misfolded form of theprotein.

In one of its aspects, the present invention provides a method fordetecting senescent cells, in which a binding agent that bindsselectively to a misfolded surface protein, i.e., the marker protein, iscombined with a sample containing cells to be examined, and determiningwhether the cells are bound by the binding agent to form a complex, thepresence of such complex indicating that the sample contains senescentcells.

The assay method can be performed using techniques such as flowcytometry, ELISA, Western blot and the like. The assay can be practisedin vitro, and in vivo by way of imaging to reveal senescent cellsendogenous to a subject.

In another of its aspects, the present invention provides a methoduseful to deplete senescent cells endogenous to a subject and/or totreat an age-related disease in a subject in need thereof, comprisingadministering to the subject a binding agent that is selectively toxicto senescent cells in an amount effective to reduce the number of suchcells, wherein the binding agent binds selectively to a senescent cellsurface protein having a misfolded conformation.

In embodiments, the binding agent is an antibody or fragment thereofthat binds selectively to the marker protein. Suitable binding agentsare agents that bind the marker protein at least two fold moreefficiently than its native protein counterpart. Particularly suitablebinding agents are antibodies, and fragments thereof that bind themarker protein with comparable efficiency. Also suitable are antibodyconjugates in which the antibody or binding fragment is conjugated witha toxin useful to kill the targeted senescent cells.

In a further aspect, the present invention provides a method useful todeplete senescent cells endogenous to a subject in need thereof and/orto treat an age-related disease in a subject in need thereof, comprisingadministering to the subject an immunogen, such as in the form of avaccine, that elicits an antibody that is selectively toxic to senescentcells, wherein the antibody binds selectively to a senescent cellsurface protein having a misfolded conformation.

In one specific embodiment, the marker protein is a misfolded form ofprion protein, PrP. In other embodiments, the marker protein is amisfolded form of a cell surface protein such as Fas receptor (FasR). Infurther embodiments, the marker protein is a misfolded form of CD38,Notch-1, CD44, CD59, Fas ligand, TNF receptor, or EGF receptor.

In embodiments, the present method exploits particularly an antibodythat binds selectively to a misfolded form of human FasR, as well asmisfolded FasR-binding antibody fragments and conjugates thereof, andpharmaceutical compositions and medical uses thereof. In addition, thereis provided a method for producing the antibody, and immunogens/vaccinesthat elicit endogenous production of the antibody.

These and other aspects of the invention are now described in greaterdetail with reference to the accompanying drawings in which:

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows gating of live cells by flow cytometry based on forwardscatter (FSC), side scatter (SSC), and viability dye exclusion(PerCP-Cy5.5).

FIG. 2 shows the binding of isotype control antibodies (mIgG and rIgG,mouse IgG and rabbit IgG, respectively), secondary antibody alone (2°Ab), the pan-PrP specific antibody 6D11, and the misfolding specificanti-PrP antibody AMF-1c-20 (DSE) to untreated and mitomycin c-treatedsenescent HUVEC cells. Analysis is by flow cytometry.

FIG. 3 shows gating of live cells by flow cytometry based on forwardscatter (FSC), side scatter (SSC), and 7AAD viability dye exclusion(PerCP-Cy5.5).

FIG. 4 shows binding isotype control antibodies (mIgG and rIgG, mouseIgG and rabbit IgG, respectively), secondary antibody alone (2° anti-mand 2° anti-r), the pan-PrP specific antibody 6D11, and the misfoldingspecific anti-Fas antibodies AMF-3a-118 and AMF-3d-19 to untreated (A)and mitocmycin c-treated senescent (B) HUVEC cells. Analysis is by flowcytometry.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery of a senescent cell marker thatcan be targeted for diagnostic and medical intervention. The marker is acell surface protein that has adopted a misfolded conformation on cellsthat are senescent. The marker is thus related to a protein found in anatively folded state on the surface of a normal, healthy cell. When thecell has entered a senescent state, however, the marker protein ismisfolded, and presents regions or domains that are not found oraccessible in the natively folded protein. These unique regions ordomains serve as epitopes for the raising of antibodies and selection ofother binding agents that, in turn, bind uniquely and selectively to themisfolded protein.

The present methods are applied diagnostically to detect senescent cellspresent in any given sample, including biological fluids or tissuesobtained from a subject, or to detect senescent cells in situ, i.e.,senescent cells that are endogenous to a subject.

The present methods are also applied therapeutically to depletesenescent cells endogenous to a subject in need of treatment. Senescentcell “depletion” refers simply to a reduction in the number of viablesenescent cells. Without wishing to be bound by theory, the result ofeliminating or depleting senescent cells is to reduce the effect ofthese cells on their neighbouring environment, such as to reduce theso-called senescence-associated secretory phenotype (SASP) effectthrough which senescent cells create a toxic local environment thataffects the health of neighbouring cells and promotes cell aging andassociated medical conditions, including tumourigenesis.

The invention is thus based on the principle that misfolded proteins arepresent on the surface of senescent cells, and that senescent cells cantherefore be identified and their deleterious effects controlled,reduced or eradicated by targeting the misfolded form of that surfaceprotein. In one approach, senescent cells are screened to identifysurface proteins that have adopted a conformation different from normalcells that present the protein in natively folded state. Onceidentified, the surface protein can be examined in silico usingestablished algorithms to identify misfolding “hot spots” within itssequence. One such algorithm is described by Cashman et al in WO2010/040209, incorporated herein by reference. As described in thatpublication, a wide variety of cell surface proteins have been examinedand epitopes that are uniquely presented when that protein misfolds areidentified. These epitopes are particularly useful targets for themethods of the present invention.

Senescent cell surface protein epitopes that are presented “uniquely” bythe misfolded form of a protein, relative to the natively folded form ofthat protein, thus provide a basis for distinguishing between the twoforms of the protein, and allow the misfolded protein to be targetedwhile substantially removing the natively folded protein as acomplicating factor in therapeutic and diagnostic applications. Inprinciple, an epitope is unique to a misfolded protein if an antibody orother ligand that binds that epitope shows significantly reduced bindingto the natively folded form of that protein. In an embodiment, thebinding difference is effective to yield a discernible test result ortherapeutic result. The misfolded form of a protein is characterized, inthe present context, as a form that presents an epitope not found in thenatively folded protein. This results from the protein adopting adifferent conformation, owing for example to different environmentalconditions (for example, oxidation of cysteine), defects in proteinfolding surveillance (for example, ineffective chaperone function),aberrant post-translational modifications (for example, defectivematuration of glycosylation), abnormal interaction with another protein(for example, PrPC/PrPSc interaction), or from genetic mutation. As aresult of misfolding, the protein can adopt an unusual and undesirablestate, with a different immunogenicity relative to its normal state.

Accordingly, an aspect of the invention includes a method to depletesenescent cells endogenous to a subject, comprising administering to thesubject a binding agent that is selectively toxic to senescent cells inan amount effective to reduce the number of such cells, wherein thebinding agent binds selectively to a senescent cell surface proteinhaving a misfolded conformation.

The term “selectively toxic to senescent cells” means that the bindingagent is at least 2 times more toxic to a senescent cell expressing themisfolded protein, compared to a cell such as a normal not expressingthe misfolded protein, such as 3 times greater, 5 times greater or atleast one order of magnitude greater (e.g., at least 2, 3, 4 or 5 ordersof magnitude greater). For instance, the cell toxicity of an antibodythat binds misfolded PrP on a senescent cell is preferably at leasttwice its toxicity for natively folded PrP on normal tissue.

Another aspect includes a method to treat an age-related disease in asubject in need thereof, comprising administering to the subject abinding agent that binds selectively to a senescent cell surface proteinhaving a misfolded conformation.

The binding agent binds selectively to an epitope exposed in a misfoldedconformation of the cell surface protein present on the senescent cell.The term “epitope” refers a region of a protein that is recognized by aB cell or T-cell receptor, or an antibody or a binding fragment thereofand is represented herein by linear peptides having amino acid sequencesthat are found in the parent protein in which the epitope resides. Theepitope will usually comprise at least 3 contiguous amino acid residues,representing the minimal domain necessary for antibody binding. However,the epitope can include a larger number of contiguous residues, such as5 residues, 7 residues which typically is the length required forminimal immunogenicity within common antibody production hosts, 10residues, 15 residues, 20 residues or more. The criticality lies inproviding an epitope that harbours an antibody-binding domain. Thus, andwith reference to Tables 1, 2 and 3, infra, it is to be appreciated thatthe peptides specifically listed, in addition to serving per se asepitopes, may further comprise additional amino acid residues at one orboth flanks thereof, particularly for those peptides that consist offewer than 7 residues. The additional residues can be those normallyassociated with the peptide in the context of the parent protein.Conversely, it should be appreciated that truncated forms of thepeptides can also serve as epitopes, especially when the peptideconsists of more than 7 residues. The extent of truncation can varydepending on the actual length of a given peptide. Any contiguous 7residues within a listed peptide can be expected to be immunogenic perse, and any 3, 4, 5, 6, or 7 contiguous residues or more can be expectedto be useful as an epitope to which antibodies can bind.

The senescent cell marker can be any surface protein that is misfoldedon the surface of a senescent cell, but is otherwise natively folded ona normal cell counterpart. Of course, the variety of surface proteinsthat are candidates as senescent cell markers are numerous. Thedetermination that a given surface protein is a senescent cell markercan be made by comparing the conformations of that protein on a normalcell vs. a senescent cell of the same type. In embodiments, the surfaceprotein is selected from prion protein (PrP), FasR, Fas ligand, CD44,EGF receptor, CD38, Notch-1, CD44, CD59, or TNF receptor, and the like.

Prion Protein, PrP, as Target

In embodiments of the present invention, the senescent cell marker is amisfolded form of prion protein, or PrP. In mature form, the human prionprotein (hPrPC) is a 230 residue protein having the sequence set out inUniProtKB/SwissProt designation P04156. This protein is known to adopt amisfolded conformation when associated with neurodegenerative diseasesincluding CJD, and when presented on the surface of certain cancercells, including particularly ovarian cancer cells. Accordingly, variousantibodies specific for the misfolded forms of PrP have been developedand are described in the literature. These antibodies and vaccines thatproduce them are useful in the present invention.

More particularly, the present invention exploits agents that binduniquely to misfolded PrP, and particularly, in embodiments of thepresent invention, to an epitope comprised by any one of the followingpeptides:

TABLE 1 SEQ Protein Target (abbr) Residue ID UniProtKB/SwissProt # spanSequence No. Human prion protein 125-130 LGGYML 1 (hPrP) P04156 126-132GGYMLGS 2 residues 1-230 140-147 HFGSDYED 3 143-147 SDYED 4 148-155RYYRENMH 5 151-155 RENMH 6 160-166 QVYYRPM 7 165-174 PMDEYSNQNN 8166-174 MDEYSNQNN 9 185-197 KQHTVTTTTKGEN 10 70-80 ARDCTVNGDEP 11105-111 RLCDEGH 12 136-142 NSTVCEH 13 167-189 EEPSRSNLGWLCL 14

In a particular embodiment, the binding agent binds selectively to anepitope that comprises a YYR motif. As shown in Table 1, the YYR motiflies within PrP residues 160-166 (SEQ ID No.7) and within PrP residues148-155 (SEQ ID No.5). These peptides and the epitopes therein areaccordingly useful targets for the binding agent. It will be appreciatedthat variations of these specific peptides that also comprise YYR andare based also on the PrP protein sequence are included as usefultargets for the binding agent. Such peptides will typically include atleast 5 contiguous residues, for instance, RYYRE (SEQ ID No.15), RYYREN(SEQ ID No.16), RYYRENM (SEQ ID No.17), DRYYRENMH (SEQ ID No.18),DRYYRENM (SEQ ID No. 19), and VYYRPM (SEQ ID No.20), QVYYRP (SEQ IDNo.21), QVYYR (SEQ ID No.22), QVYYRPMD (SEQ ID No.23), and the like.

In another embodiment, the binding agent is one that binds selectivelyto a misfolded PrP epitope that comprises a YML motif. This particularmotif lies within PrP beta strand 1. As shown in Table 1 above, the YMLmotif is common within PrP peptides, LGGYML and GGYMLGS (SEQ ID Nos. 1and 2, respectively). These peptides are accordingly also useful epitopetargets for the binding agent. It will be appreciated that variations ofthese specific peptides that also comprise YML and are based also onparent protein sequence are included as useful epitope targets inembodiments of the present invention. Such peptides will typicallyinclude at least 3, 4 or more usually at least 5 contiguous residues,for instance, GGYMLG (SEQ ID NO: 25), GYMLGS (SEQ ID NO: 26), GGYML (SEQID NO: 27), YMLGS (SEQ ID NO: 28), GYML (SEQ ID NO: 29), YMLG (SEQ IDNO: 30) LGGYML (SEQ ID No. 31), LGGYMLG (SEQ ID No. 32) and YML (SEQ IDNO: 33).

In a further embodiment, the binding agent binds selectively to amisfolded PrP epitope that comprises a MDEYSNQNN motif. This particularmotif, located between β-sheet 2 and α-helix 1, is known also as therigid loop, and lies within PrP residues 167-174, as shown in Table 1.It will be appreciated that variations of these specific peptides thatalso comprise at least about 5 contiguous residues within the rigid loopsequence and are based also on parent protein sequence are included asuseful epitope targets in embodiments of the present invention. Suchpeptides will typically include at least 5 contiguous residues, forinstance, PMDEYSNQNN (SEQ ID No.34), DEYSNQNN (SEQ ID No.35), andMDEYSNQ (SEQ ID No.36).

Agents that bind to these epitopes will bind selectively to misfoldedPrP, relative to natively folded PrP, and will thus bind selectively tosenescent cells in which surface PrP is misfolded, relative to normalcells.

Agents that bind to these epitopes can be identified using routinescreening methods in which a panel of putative ligands is combined witha peptide presenting the epitope, and binding events are then analyzedto identify peptide binding partners. Ligands that also bind to PrP inits wild type, natively folded state can be dismissed, so that only theligands or binding partners that bind uniquely to the epitope targetsare selected.

It will be appreciated that the binding agents useful herein can takethe form of small molecules, peptides, peptidomimetics, peptide ornucleic acid aptamers, and the like. In particular embodiments, thebinding agent is an antibody or an epitope-binding fragment thereof. Inother embodiments, the binding agent is induced by the administration ofan immunogen that elicits production within the recipient of antibodiesthat bind the senescent cell marker.

In embodiments, the binding agent is an antibody that binds selectivelyto PrP in a misfolded form presented by senescent cells. In particularembodiments, the antibody is one that binds selectively to a peptidelisted in Table 1.

In more specific embodiments, the antibody is one that binds a peptideincorporating the YML motif of PrP. Particularly, the antibody is theantibody designated 1A1, as described in more detail in WO 2010/099612incorporated herein by reference. This antibody displays selectivebinding to the prion protein YML motif. Its hybridoma source wasdeposited under terms of the Budapest Treat at the InternationalDepositary Authority of Canada (IDAC) under accession number 260210-01.

In other specific embodiments, the antibody is one that binds a peptideincorporating the YYR motif of PrP. Particularly, the antibody is onethat is raised in the manner described in WO 03/000853 (and see U.S.Pat. No. 7,041,807) incorporated herein by reference, and as reproducedin Example 1 herein for convenience.

In another specific embodiment, the antibody is one that binds the rigidloop of PrP. Particularly, the antibody is the antibody designatedc-120, as described in co-pending WO2013/185215 published on 2013 Dec.19, and incorporated herein by reference. This antibody is defined byheavy and light chains having the sequences set out in SEQ ID No.38 andSEQ ID No. 37, respectively.

The variable regions of the antibody have the sequences set out in SEQID No.39 for the light chain variable region, and in SEQ ID No.40 forthe heavy chain variable region.

The sequences of the important complementarity determining regions ofthe c-120 antibody have the following sequences:

For the heavy chain:

(SEQ ID No. 41) CDR1 TYAMG (SEQ ID No. 42) CDR2 VITKSGNTYYASWAKG(SEQ ID No. 43) CDR3 YGIGVSYYDI

For the light chain:

(SEQ ID No. 44) CDR1 QSSQSLYNKNWLS (SEQ ID No. 45) CDR2 KASTLES(SEQ ID No. 46) CDR3 QGEFSCSSADCTA

It will be appreciated that the binding agent thus can be an antibody ora fragment thereof that binds selectively to the rigid loop of PrP andincorporates at least the CDR sequences provided above.

Cell surface proteins other than PrP are also misfolded when the hostcell is in a state of senescence. Like PrP, the conformationalalteration results in the presentation of new epitopes that are uniqueto the misfolded protein, relative to its native counterpart. Theseepitopes are useful targets for senescent cell detection and depletion.More particularly, the present inventors have applied an algorithm thatpredicts misfolding “hot spots” within surface proteins. That algorithmis described by Cashman et al in WO 2010/040209, and was used toidentify the misfolding specific epitopes within the target surfaceproteins shown below.

TABLE 2 Protein Target (abbr) SEQ UniProtKB/SwissProt Residue ID # spanSequence No. Human CD44 P16070 39-45 NGRYSIS 47 residues 21-742 83-87EGHVV 48 108-116 TSNTSNYDT 49 149-159 NRDGTRYVQKG 50  99-116ANNTFVYILTSNTSNYDT 51 Human tumour 21-31 IHPQNNSICCT 52 necrosis 41-51NDCPGPGQDTD 53 factor receptor 106-112 YWSENLF 54 (hTNFR)P19438 126-134HLSCQEKQN 55 139-151 CTCHAGFFLRENECV 56 Human notch protein 423-429CEHAGKC 57 (hNOTCH1)P46531 479-487 MPGYEGVHC 58 residues 1-2550 499-505CLHNGRC 59 508-512 KINEF 60 493-517 ECASSPCLHNGRCLDKIN 61 EFQCECPHuman Fas receptor 52-60 LHHDGQFCH 62 P25445 residues 26- 70-80ARDCTVNGDEP 63 335 105-111 RLCDEGH 64 136-142 NSTVCEH 65 167-189EEGSRSNLGWLCL 66 Human epidermal 11-25 SNKLTQLFTFEDHFL 67 growth factor46-56 VQRNYDLSFLK 68 receptor 83-95 IRGNMYYENSYAL 69 (hEGFR)P00533 97-107 VLSNYDANKTG 70 residues 25-1210 143-155 IVSSDFLSNMSMD 71 148-169FLSNMSMDFQNHLGS 72 176-181 WGAGEE 73 241-259 PPLMLYNPTTYQMDVNPE 74257-266 PEGKYSFGAT 75 273-282 RNYVVTDHGS 76 288-301 GADSYEMEEDGVRK 77314-328 NGIGIGEFKDSLSIN 78 328-337 ATNIKHFKN 79 348-363 LPVAFRGDSFTHTPPL80 465-474 KIISNRGENS 81 Human CD38 70-84 YTEIHPEMRHVDCQS 82P28907 1-300 161-169 GEFATSKIN 83 176-182 WRKDCSN 84 210-220 GSRSKIFDKDS85 243-253 IHGGREDSRDL 86

It has been determined further that the cell surface protein human FasR(Fas receptor) is also misfolded when present on senescent cells. Fasreceptor (FasR) is known also as human tumour necrosis factorsuperfamily member 6 receptor (hTNFRSM6), and as CD95, and is implicatedin cancer. It is a death receptor on the surface of cells that leads tocaspase-mediated programmed cell death (apoptosis). More particularly,the present invention provides and exploits agents that bind uniquely tomisfolded FasR, and particularly, in embodiments of the presentinvention, to an epitope comprised by any one of the peptides shown inTable 3:

TABLE 3 Human FasR 52-60 LHHDGQFCH 62 (hTNFRSM6) 70-80 ARDCTVNGDEP 63P25445 residues 26- 105-111 RLCDEGH 64 335 136-142 NSTVCEH 65 167-189EEPSRSNLGWLCL 66

In a particular embodiment, the binding agent binds selectively to aFasR epitope that comprises an LHHDGQFCH sequence (SEQ ID No.62). In analternative embodiment, the binding agent binds selectively to a FasRepitope that comprises an NSTVCEH sequence (SEQ ID No.65).

Also provided herein as binding agents that bind selectively tomisfolded FasR, are antibodies that bind to one of the two peptidesequences just recited. In particular, and in one of its aspects, thepresent invention provides an antibody designated AMF 3a-118, theantibody having been raised against the LHHDGQFCH in the mannerexemplified herein. The heavy chain of the 3a-118 antibody has SEQ IDNo. 89. The light chain of the 3a-118 antibody has SEQ ID No. 91. Theheavy chain variable region of the 3a-118 antibody has SEQ ID No. 90.The light chain variable region of the 3a-118 antibody has SEQ ID No.92. The misfolded FasR binding site presented by this antibody comprisesthe following CDRs:

For the heavy chain

(SEQ ID No. 93) CDR1 DSRVS (SEQ ID No. 94) CDR2 IVGIGWNIYHANWAKG(SEQ ID No. 95) CDR3 GLGGGTVI

For the light chain

(SEQ ID No. 96) CDR1 QSSESVYKNNYLS (SEQ ID No. 97) CDR2 EASKLAS(SEQ ID No. 98) CDR3 LGEFSCYSGDCGT

In addition, the present invention provides an antibody designated AMF3d-19, the antibody having been raised against the NSTVCEH sequence inthe manner exemplified herein. The heavy chain of the 3d-19 antibody hasSEQ ID No. 103. The light chain of the 3d-19 antibody has SEQ ID No.105. The heavy chain variable region of the 3d-19 antibody has SEQ IDNo. 104. The light chain variable region of the 3d-19 antibody has SEQID No. 106. The misfolded FasR binding site presented by this antibodycomprises the following CDRs:

For the heavy chain

(SEQ ID No. 107) CDR1 RNAIN (SEQ ID No. 108) CDR2 IIGSSGVTYYASWAKG(SEQ ID No. 109) CDR3 NLYTGGSNDNL

For the light chain

(SEQ ID No. 110) CDR1 QASKSVYNNVQLS (SEQ ID No. 111) CDR2 YASTLAS(SEQ ID No. 112) CDR3 AGGYSSSSDNA

It will thus be appreciated that the binding agent useful in the presentmethods can be any antibody or a fragment thereof that binds selectivelyto a protein that is misfolded on the surface of a senescent cell, bybinding to a particular epitope that is unique to that misfolded formrelative to the natively folded protein.

The antibodies that bind selectively to the misfolding specific epitopesmay be either polyclonal or monoclonal, of the IgG or IgM class, and maybe derived from any mammal, particularly goats, rabbits or mice, or byrecombinant methods. More generally, it will be appreciated thatantibodies useful in the present invention include the various intactforms including polyclonal antibodies, monoclonal antibodies, andrecombinant antibodies including chimeric antibodies, humanizedantibodies as well as fully human antibodies and bispecific ormultispecific antibodies.

In an embodiment, the antibody comprises a human constant region.

The chimeric antibodies comprise a portion of the heavy and/or lightchain that is homologous with corresponding sequences in antibodiesderived from a particular species, or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is homologouswith corresponding sequences derived from another species or belongingto a different antibody class. Humanized antibodies are chimericantibodies that comprise minimal sequence derived from non-humanantibody, usually incorporating CDRs from a non-human antibody into ahuman antibody framework, which may further be altered to incorporatenon-human residues that restore and enhance antigen binding. The “fully”human antibodies can be produced in a non-human host using varioustechniques that are now established, including through the use of phagedisplay libraries, and particularly by introducing human immunoglobulinloci into transgenic animals such as mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibodies are produced which closely resemble thatseen in humans in most respects, including gene rearrangement, assemblyand antibody repertoire.

Using phage display antibodies are displayed on the surface of phage byfor example fusing the coding sequence of antibody variable regions tothe phage minor coat protein pill. Antibodies can be selected using thephage displayed antibody libraries (including synthetic antibodylibraries wherein synthetic diversity is introduced at solvent-exposedpositions within the heavy chain complementarity-determining regions) bya series of cycles of selection on antigen. Antibody genes can be clonedsimultaneously with selection and further engineered for example byincreasing their affinity or modulating their specificity or theireffector function (by recloning into a full-length immunoglobulinscaffold e.g. making a recombinant human antibody).

In an embodiment, the antibody is a recombinant or synthetic antibody,for example the human antibody is a recombinant or synthetic humanantibody.

The antibodies may be of any useful class, including IgA, IgD, IgE, IgGand IgM, and isotypes including IgG1, IgG2, IgG3, and IgG4. The constantregion (Fc) of the antibodies can also be engineered or conjugated toprovide altered effector function, thereby to enhance antibody dependentcell-mediated cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) activity.

For therapeutic use, in embodiments, the antibody desirably is itselftoxic to the senescent cells presenting the target epitope. To this end,the antibody desirably incorporates an Fc region having effectorfunction, such as ADCC activity and/or CDC activity. In the alternative,and in accordance with other embodiments, the antibody or bindingfragment is conjugated with a cytotoxin that is toxic to the senescentcells targeted by the antibody or binding fragment.

The antibodies otherwise can have all of the attributes common to intactantibodies. In embodiments, the present antibodies are of the IgG1isotype, but they may also be IgG2 or IgG4. Moreover, the isotype of theantibody, as dictated by the constant region, can be manipulated toalter or eliminate the effector function of the resulting antibody. Thatis, the constant region of the present antibodies is either wild typehuman antibody constant region, or a variant thereof that incorporatesamino acid modifications, i.e., amino acid additions, substitutions ordeletions that alter the effector function of the constant region, suchas to enhance serum half-life, reduce or enhance complement fixation,reduce or enhance antigen-dependent cellular cytotoxicity and improveantibody stability. The number of amino acid modifications in theconstant region is usually not more than 20, such as 1-10 e.g., 1-5modifications, including conservative amino acid substitutions.

In embodiments, the half-life of the antibody is improved byincorporating one or more amino acid modifications, usually in the formof amino acid substitutions, for instance at residue 252, e.g., tointroduce Thr, at residue 254, e.g., to introduce Ser, and/or at residue256 e.g., to introduce Phe. Still other modifications can be made toimprove half-life, such as by altering the CH1 or CL region to introducea salvage receptor motif, such as that found in the two loops of a CH2domain of an Fc region of an IgG. Such alterations are described forinstance in U.S. Pat. No. 5,869,046 and U.S. Pat. No. 6,121,022.

Altered C1 q binding, or reduced complement dependent cytotoxicity, canbe introduced by altering constant region amino acids at locations 329,331 and 322, as described in U.S. Pat. No. 6,194,551. The ability of theantibody to fix complement can further be altered by introducingsubstitutions at positions 231 and 239 of the constant region, asdescribed in WO94/029351.

Framework modifications can also be made to reduce immunogenicity of theantibody or to reduce or remove T cell epitopes that reside therein, asdescribed for instance by Carr et al in US2003/0153043.

Antibodies can also be altered in the variable region to eliminate oneor more glycosylation sites, and/or to improve physical stability of theantibody. For example, in one embodiment, the physical stability of theantibody is improved by substituting the serine at position 228 of thevariable region with a proline residue (i.e., the antibody has avariable region comprising a S228P mutation). The S228P alterationsignificantly stabilizes the antibody structure against the formation ofintrachain disulfide bonds. In another embodiment, the variable regionis altered to eliminate one or more glycosylation sites resident in thevariable region. More particularly, it is desirable in the sequence ofthe present antibodies to eliminate sites prone to glycosylation. Thisis achieved by altering the occurrence of one or more N-X-(S/T)sequences that occur in the parent variable region (where X is any aminoacid residue), particularly by substituting the N residue and/or the Sor T residue.

Antibodies can be engineered to include a variety of constant regions.In one embodiment, the antibody comprises a constant region the sequenceof which corresponds to the constant region of an antibody of humanorigin, such as a human IgG1 constant region. In a particularembodiment, the constant region is inert for effector function (e.g.,essentially devoid of effector function). In a specific embodiment theconstant region is a human IgG4 constant region.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072. Moreover,the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutationsat positions 256, 290, 298, 333, 334 and 339 were shown to improvebinding to FcγRIII. Additionally, the following combination mutants wereshown to improve FcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224Aand S298A/E333A/K334A.

In addition, the antibody can be pegylated, for example, to increase thebiological (e.g., serum) half-life of the antibody. To pegylate anantibody, the antibody, or fragment thereof, typically is reacted withpolyethylene glycol (PEG), such as a reactive ester or aldehydederivative of PEG, under conditions in which one or more PEG groupsbecome attached to the antibody or antibody fragment. Preferably, thepegylation is carried out via an acylation reaction or an alkylationreaction with a reactive PEG molecule (or an analogous reactivewater-soluble polymer). As used herein, the term “polyethylene glycol”is intended to encompass any of the forms of PEG that have been used toderivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See, e.g., EP 154316 and EP 401384.

The antibodies and binding fragments are useful both for diagnosticpurposes, including in vivo imaging to identify endogenous sites ofsenescent cells in vivo, and for sample testing to detect senescentcells in vitro. The antibodies and binding fragments are also useful fortherapeutic purposes to treat diseases in which senescent cells areimplicated.

“Selective-binding” agents are agents that bind the target epitope, andbind proteins that present the target epitope in a solvent-accessibleorientation, with an affinity that is at least 2 times greater than theaffinity with which they bind a different, unrelated epitope, such as 3times greater, 5 times greater or at least one order of magnitudegreater (e.g., at least 2, 3, 4 or 5 orders of magnitude greater Forinstance, the binding affinity of an antibody that binds misfolded PrPon a senescent cell is preferably at least twice its binding affinityfor natively folded PrP on normal tissue. Relative binding affinitiescan be determined, and the antibody so selected, on the basis of assaysand techniques that generally are well established in the art for thispurpose.

In an embodiment, the antibody affinity has an EC50 which is at least10⁻⁶ M, at least 10⁻⁷ M, at least 10⁻⁸ M, or at least 10⁻⁹ M.

All antibodies were then tested by ELISA for binding to denaturedfull-length recombinant PrP. One antibody exhibited a titer fordenatured PrP that was similar to the anti-peptide affinities, in the10⁻¹⁰ M range. Thus, the preferred antibody exhibits preferably an EC50by this test that is at least better than 10⁻⁹M.

Antibodies that bind selectively to the epitope unique to a misfoldedform of a protein can be produced by techniques including immunization,or by alternative approaches such as by the application of phage displayand other systems that use high throughput to identify complementaritydetermining regions (CDR) or other sequences that bind to the targetepitope. It is not essential that the resulting antibody has been firstraised in vivo. More particularly, to produce suitable antibodies, aminoacid sequences that constitute epitopes can be useful per se to raiseantibodies that bind specifically to them, provided they are endowed perse with the immunogenicity required to raise antibody in the selectedantibody production host. For those epitopes that lack suchimmunogenicity, it is desirable to provide an immunogen that containsthe epitope sequence.

As used herein, “immunogen” refers to an immunogenic form of a peptideor other molecule that comprises the epitope, and is represented by thepeptide itself when immunogenic per se, or is represented by the peptidein combination with an immunogenicity-enhancing agent. Any of theestablished agents can be used for this purpose. These agents typicallyinclude carrier proteins that can be coupled to the epitope eitherdirectly, such as through an amide bond, or indirectly through achemical linker such as carbodiimide, a cysteine, or any peptide spacersequence such as a glycine or glycine-serine sequence including Gly4-S.For example, an isolated peptide comprising a given epitope can beconjugated to MAP antigen, OVA antigen, or keyhole limpet hemocyanin(KLH). Its large size makes it very immunogenic, and the large number oflysine residues available for conjugation makes KLH very useful toattach to a polypeptide. The immunogen may further comprise a linkereffective to couple the peptide tandemly to another copy of the same ora different peptide corresponding to the same or a different epitope. Inanother embodiment, the peptides may comprise additional amino acidsthat enhance the immunogenicity or solubility of the peptide. In oneembodiment, the additional amino acids number from 1 to about 10,preferably 1 to 8, more preferably 1 to 5. Importantly the additionalresidues do not materially affect the conformation of the peptide.

Thus, for antibody production, epitopes that are not themselvesimmunogenic and do not constitute an immunogen can be rendered so, andprovided as an immunogen, by incorporating immune enhancing agents thatare either conjugated therewith or coupled covalently.

A composition comprising the immunogen can be prepared for purposes ofproducing antibodies in a selected host by combining the immunogen withan appropriate vehicle. Such vehicles include Freund's complete adjuvantor other adjuvant or a suitable saline or phosphate buffered salinesolution (0.05-1.0%).

Antibodies are then prepared to react against these epitopes when theyare in an unstructured state. As noted, each peptide may be conjugatedto a carrier protein like KLH to form an immunogen that is injected,optionally in combination with an adjuvant such as Freund's completeadjuvant, into a mammalian production host like a mouse, rat, rabbit,sheep or goat to provoke an immune response that generates antibodiesagainst the peptide. Standard immunization protocols can be used, andthe antibodies can be recovered from blood by enrichment against theimmunizing agent, as exemplified herein.

The antibodies useful herein are desirably “isolated” antibodies, whichrefers to an antibody that is substantially free of other antibodieshaving different antigenic specificities (e.g., an isolated antibodythat specifically binds misfolded PrP is substantially free ofantibodies that specifically bind antigens other than PrP proteins). Anisolated antibody that specifically binds a misfolded human PrP proteinmay, however, have cross-reactivity to other antigens, such as misfoldedPrP proteins from other species. Moreover, an isolated antibody can besubstantially free of other cellular material and/or chemicals. Anisolated antibody also can be substantially free of other proteins ofhuman origin. In embodiments, the isolated antibody is an exogenousantibody as distinct from an antibody endogenous to an intendedrecipient.

Thus, in embodiments, the antibody is an intact antibody comprisingfeatures common to all natural antibodies, e.g., a heavy chain and alight chain, each chain having a constant region and a variable region,each variable region comprising framework regions (FRs) andcomplementarity determining regions (CDRs). In the alternative, theantibody is provided as a target-binding fragment, such as a monovalentfragment, Fab, or a bivalent antibody fragment comprising both “arms” ofan intact antibody, joined through a linker that can be represented bythe hinge region of the antibody or any equivalent. Such fragmentsinclude F(ab)2 fragments and any other fragment that retains preferencefor binding to misfolded PrP. In particular embodiments, the antibodyfragment is a F(ab′)2 fragment, generated for instance by papain-baseddigestion of the parent antibody using standard procedures for digestionand subsequent fragment isolation. In the alternative, the fragment canbe a so-called single chain Fv (scFv), consisting of the variable lightand variable heavy antibody domains joined by an amino acid linker, or abivalent form of a so-called diabody prepared using a 5 amino acidlinker such as SGGGG (SEQ ID NO: 117) between the light and heavy chainvariable domains and a C-terminal cysteine modification to GGC to give afinal diabody product as VL-SGGG-VH-GGC. Still other bivalent fragmentscan be prepared by coupling the light and heavy chain variable domainsthrough thioether linkages such as bis-maleimidomethyl ether (BMME),N,N′-p-phenylene dimaleimide (PDM and N,N′-bismaleimidohexane BMH), tostabilize the F(ab′)2 fragments.

Of course, for antibodies having known protein or gene sequences, theantibody can be produced suitably by recombinant DNA means. Forproduction, there is provided a DNA molecule that encodes the heavychain of the present antibody, and a DNA molecule that encodes the lightchain thereof. The DNA further encodes any suitable signal peptidesuitable for expression of a secretable chain precursor that enablesproper externalization with folding and disulfide formation to elaboratethe desired antibody as a secreted, dimerized and processed protein.

To this end, the present invention provides, in one aspect,polynucleotides that encode the heavy and light chains of the FasRantibodies herein described. In one embodiment, there is provided apolynucleotide comprising a sequence that encodes the light chainvariable region of the FasR antibody AMF 3a-118, as set out in SEQ IDNo.102. Also provided, in another embodiment, is a polynucleotidecomprising a sequence that encodes the heavy chain variable region ofthe FasR antibody AMF 3a-118, as set out in SEQ ID No.100.

In more specific embodiments, the present invention provides apolynucleotide that encodes the entire light chain (SEQ ID No. 101) anda polynucleotide that encodes the entire heavy chain (SEQ ID No. 99) ofmisfolded FasR antibody AMF 3a-118 antibody.

The present invention also provides, in another aspect, polynucleotidesthat encode the light chain variable region of the FasR antibody AMF3d-19, as set out in SEQ ID No. 116, and a polynucleotide comprising asequence that encodes the heavy chain variable region of the FasRantibody AMF 3d-19, as set out in SEQ ID No. 114.

In more specific embodiments, the present invention provides apolynucleotide that encodes the entire light chain (SEQ ID No. 115) anda polynucleotide that encodes the entire heavy chain (SEQ ID No. 113) ofthe misfolded FasR antibody AMF 3d-19

It will be appreciated that polynucleotide equivalents also can be used,in which synonymous codons are replaced within the sequences provided,to produce the present antibodies. In an embodiment, the nucleic acid isa cDNA. In another embodiment, the nucleic acid is a codon optimizedcDNA.

In embodiments, there are also provided vectors that comprisepolynucleotides that encode the heavy chain or the variable regionthereof and that encode the light chain or the variable region thereof.To express the antibodies, the polynucleotides are incorporated operablywithin expression vectors, i.e. operatively linked to transcriptionaland translational control sequences. Expression vectors includeplasmids, retroviruses, cosmids, and the like. The expression vector andexpression control sequences are chosen to be compatible with theexpression host cell used. The antibody light chain gene and theantibody heavy gene can be inserted into separate vectors. In apreferred embodiment, both genes are inserted into the same expressionvector. The antibody genes are inserted into the expression vector bystandard methods (e.g., ligation of complementary restriction sites onthe antibody gene fragment and vector, or blunt end ligation if norestriction sites are present).

A convenient vector is one that encodes a functionally complete human CHor CL immunoglobulin sequence, with appropriate restriction sitesengineered so that any VH or VL sequence can be easily inserted andexpressed, as described above. In such vectors, splicing usually occursbetween the splice donor site in the inserted J region, and the spliceacceptor site preceding the human C region, and also at the spliceregions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The recombinant expression vector can also encodea signal peptide that facilitates secretion of the antibody chain from ahost cell. The antibody chain gene may be cloned into the vector suchthat the signal peptide is linked in-frame to the amino terminus of theantibody chain gene. The signal peptide can be an immunoglobulin signalpeptide or a heterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

Polynucleotides encoding the heavy chain and/or the light chain, andvectors comprising these can be used for transformation of a suitablemammalian host cell. Methods for introduction of heterologouspolynucleotides into mammalian calls include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene-mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, biolistic injection and directmicroinjection of the DNA into nuclei. In addition, polynucleotides maybe introduced into mammalian cells by viral vectors.

Mammalian cell lines useful as hosts for expression of theantibody-encoding polynucleotides include many immortalized cell linesavailable from the American Type Culture Collection (ATCC). Theseinclude, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells,HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS),human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, 3T3cells, and a number of other cell lines. In a specific embodiment, thepolynucleotides are expressed in a HEK293 host. Mammalian host cellsinclude human, mouse, rat, dog, monkey, pig, goat, bovine, horse, andhamster cells. Cell lines of particular preference are selected throughdetermining which cell lines have high expression levels. Other celllines that may be used are insect cell lines, such as S19 cells,amphibian cells, bacterial cells, plant cells and fungal cells. Whenrecombinant expression vectors encoding the heavy chain orantigen-binding portion thereof are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can be,recovered from the culture medium using standard protein purificationmethods.

The antibodies of the invention can be obtained as human monoclonalantibodies. Such human monoclonal antibodies can be generated usingtransgenic or transchromosomic mice carrying parts of the human immunesystem rather than the mouse system. These transgenic andtranschromosomic mice include mice referred to herein as the HuMAbMouse® and KM Mouse®, respectively, and are collectively referred toherein as “human Ig mice.”

The HuMAb Mouse® (Medarex®, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal antibodies (Lonberg et al. (1994), supra; reviewed in Lonberg(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding andLonberg (1995) Ann. N.Y. Acad. Sci. 764:536-546). Preparation and use ofthe HuMAb Mouse®, and the genomic modifications carried by such mice, isfurther described in Taylor et al. (1992) Nucleic Acids Research20:6287-6295; Chen et al. (1993) International Immunology 5: 647-656;Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi etal. (1993) Nature Genetics 4:117-123; Chen et al. (1993) EMBO J. 12:821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor et al.(1994) International Immunology 6: 579-591; and Fishwild et al. (1996)Nature Biotechnology 14: 845-851, the contents of all of which arehereby specifically incorporated by reference in their entirety. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and5,545,807; PCT Publication Nos. WO 92/03918; WO 93/12227; WO 94/25585;WO 97/13852; WO 98/24884; WO 99/45962 and WO 01/14424, the contents ofwhich are incorporated herein by reference in their entirety.

In another embodiment, the human antibodies are raised using a mousethat carries human immunoglobulin sequences on transgenes andtranschomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. This mouse isreferred to herein as a “KM Mouse®,” and is described in detail in PCTPublication WO 02/43478. A modified form of this mouse, which furthercomprises a homozygous disruption of the endogenous FcR

IIB receptor gene, is also described in PCT Publication WO 02/43478 andreferred to herein as a “KM/FCGR2D Mouse®.” In addition, mice witheither the HCo7 or HCo12 heavy chain transgenes or both can be used.

Additional transgenic animal embodiments include the Xenomouse (Abgenix,Inc., U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and6,162,963). Further embodiments include “TC mice” (Tomizuka et al.(2000) Proc. Natl. Acad. Sci. USA 97:722-727) and cows carrying humanheavy and light chain transchromosomes (Kuroiwa et al. (2002) NatureBiotechnology 20:889-894; PCT Publication WO 02/092812). The contents ofthese patents and publications are specifically incorporated herein byreference in their entirety.

Human monoclonal antibodies also can be prepared using SCID mice intowhich human immune cells have been reconstituted such that a humanantibody response can be generated upon immunization. See, e.g., U.S.Pat. Nos. 5,476,996 and 5,698,767, the contents of which areincorporated herein by reference in their entirety.

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNAencoding partial or full-length light and heavy chains obtained bystandard molecular biology techniques is inserted into one or moreexpression vectors such that the genes are operatively linked totranscriptional and translational regulatory sequences. In this context,the term “operatively linked” is intended to mean that an antibody geneis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene.

The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals) that control the transcription or translation of the antibodychain genes. Such regulatory sequences are described, e.g., in Goeddel(Gene Expression Technology. Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990)). Preferred regulatory sequences for mammalianhost cell expression include viral elements that direct high levels ofprotein expression in mammalian cells, such as promoters and/orenhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40),adenovirus, (e.g., the adenovirus major late promoter (AdMLP) andpolyoma.

Alternatively, nonviral regulatory sequences can be used, such as theubiquitin promoter or

-globin promoter. Still further, regulatory elements composed ofsequences from different sources, such as the SRα promoter system, whichcontains sequences from the SV40 early promoter and the long terminalrepeat of human T cell leukemia virus type 1 (Takebe et al. (1988) Mol.Cell. Biol. 8:466-472). The expression vector and expression controlsequences are chosen to be compatible with the expression host cellused.

The antibody light chain gene and the antibody heavy chain gene can beinserted into the same or separate expression vectors. In preferredembodiments, the variable regions are used to create full-lengthantibody genes of any antibody isotype by inserting them into expressionvectors already encoding heavy chain constant and light chain constantregions of the desired isotype such that the VH segment is operativelylinked to the CH segment(s) within the vector and the VL segment isoperatively linked to the CL segment within the vector. Additionally oralternatively, the recombinant expression vector can encode a signalpeptide that facilitates secretion of the antibody chain from a hostcell. The antibody chain gene can be cloned into the vector such thatthe signal peptide is linked in-frame to the amino terminus of theantibody chain gene. The signal peptide can be an immunoglobulin signalpeptide or a heterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention can carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216; 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Preferred selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because mammalian cells are more likely than prokaryotic cellsto assemble and secrete a properly folded and immunologically activeantibody.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol.159:601-621), NSO myeloma cells, COS cells, HEK293 cells and SP2 cells.In particular, for use with NSO myeloma cells, another preferredexpression system is the GS gene expression system disclosed in WO87/04462, WO 89/01036 and EP 338,841. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Thus, in another aspect, there is provided a cellular host thatincorporates expressibly therein a polynucleotide that encodes the lightchain, or the light chain variable region, of the misfolded human FasRantibody designated 3a-118, the polynucleotides having SEQ ID No. 101(entire light chain) or SEQ ID No,102, respectively. In otherembodiments, there is provided a cellular host that incorporatesexpressibly therein a polynucleotide that encodes the heavy chain, orthe heavy chain variable region, of the misfolded human FasR antibodydesignated 3a-118, having SEQ ID No. 99 (entire heavy chain) or SEQ IDNo. 100 (heavy chain variable region). In a further embodiment, there isprovided a cellular host that incorporates expressibly therein both theheavy and light chain-encoding polynucleotides just recited, in eitherfully length form or in the form of variable region-encodingpolynucleotides. In a still further embodiment, there comprises the stepof culturing the transfected cellular host, thereby to produce thedesired 3a-118 antibody.

Further, in embodiments, there is provided a cellular host thatincorporates expressibly therein a polynucleotide that encodes the lightchain, or the light chain variable region, of the misfolded human FasRantibody designated 3a-118, the polynucleotides having SEQ ID No. 101(entire light chain) or SEQ ID No,102 In other embodiments, there isprovided a cellular host that incorporates expressibly therein apolynucleotide that encodes the entire heavy chain SEQ ID No.99), or theheavy chain variable region (SEQ ID No.100), of the misfolded human FasRantibody designated 3a-118. In a further embodiment, there is provided acellular host that incorporates expressibly therein both the heavy andlight chain-encoding polynucleotides just recited, in either fullylength form or in the form of variable region-encoding polynucleotides.In a still further embodiment, there comprises the step of culturing thetransfected cellular host, thereby to produce the desired 3a-118antibody.

Also, in embodiments, there is provided a cellular host thatincorporates expressibly therein a polynucleotide that encodes the lightchain, or the light chain variable region, of the misfolded human FasRantibody designated 3d-19, the polynucleotides having SEQ ID No. 115(entire light chain) or SEQ ID No,116, respectively. In otherembodiments, there is provided a cellular host that incorporatesexpressibly therein a polynucleotide that encodes the heavy chain, orthe heavy chain variable region, of the misfolded human FasR antibodydesignated 3d-19, having SEQ ID No. 113 (entire heavy chain) or SEQ IDNo. 114 (heavy chain variable region). In a further embodiment, there isprovided a cellular host that incorporates expressibly therein both theheavy and light chain-encoding polynucleotides just recited, in eitherfully length form or in the form of variable region-encodingpolynucleotides. In a still further embodiment, there comprises the stepof culturing the transfected cellular host, thereby to produce thedesired 3d-19 antibody.

Further, in embodiments, there is provided a cellular host thatincorporates expressibly therein a polynucleotide that encodes the lightchain, or the light chain variable region, of the misfolded human FasRantibody designated 3d-19, the polynucleotides having SEQ ID No. 115(entire light chain) or SEQ ID No,116 In other embodiments, there isprovided a cellular host that incorporates expressibly therein apolynucleotide that encodes the entire heavy chain SEQ ID No.113), orthe heavy chain variable region (SEQ ID No.114), of the misfolded humanFasR antibody designated 3d-19. In a further embodiment, there isprovided a cellular host that incorporates expressibly therein both theheavy and light chain-encoding polynucleotides just recited, in eitherfully length form or in the form of variable region-encodingpolynucleotides. In a still further embodiment, there comprises the stepof culturing the transfected cellular host, thereby to produce thedesired 3d-19 antibody.

For use in the methods of the present invention, the antibodies andtheir binding fragments can be conjugated with other agents that areuseful for the intended purpose, e.g., either diagnostic use or medicaltreatment. Agents appropriate for treating disease include cytotoxicagents or toxins that include chemotherapeutics and radiotherapeutics.For diagnostic purposes, appropriate agents are detectable labels thatinclude radioisotopes or fluorescent markers for whole body imaging, andradioisotopes, enzymes, fluorescent labels and the like for sampletesting.

For diagnostics, the detectable labels can be any of the various typesused currently in the field of in vitro diagnostics, includingparticulate labels including biotin/streptavidin, metal sols such ascolloidal gold, isotopes such as 1125 or Tc99 presented for instancewith a peptidic chelating agent of the N2S2, N3S or N4 type,chromophores including fluorescent markers such as FITC and PE,luminescent markers, phosphorescent markers and the like, as well asenzyme labels that convert a given substrate to a detectable marker, andpolynucleotide tags that are revealed following amplification such as bypolymerase chain reaction. Suitable enzyme labels include horseradishperoxidase, alkaline phosphatase and the like. For instance, the labelcan be the enzyme alkaline phosphatase, detected by measuring thepresence or formation of chemiluminescence following conversion of 1,2dioxetane substrates such as adamantyl methoxy phosphoryloxy phenyldioxetane (AMPPD), disodium3-(4-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo{3.3.1.13,7}decan}-4-yl)phenylphosphate (CSPD), as well as CDP and CDP-Star® or other luminescentsubstrates well-known to those in the art, for example the chelates ofsuitable lanthanides such as Terbium(III) and Europium(III). Thedetection means is determined by the chosen label. Appearance of thelabel or its reaction products can be achieved using the naked eye, inthe case where the label is particulate and accumulates at appropriatelevels, or using instruments such as a spectrophotometer, a luminometer,a fluorimeter, and the like, all in accordance with standard practice.

For therapy, the cytotoxin can be conjugated with the antibody orbinding fragment through non-covalent interaction, but more desirably,by covalent linkage either directly or, more preferably, through asuitable linker. In a preferred embodiment, the conjugate comprises acytotoxin and an antibody. Immunoconjugates of the antibody andcytotoxin are made using a variety of bifunctional protein couplingagents such as N-succinimidyl-3-(2-pyridyldithiol) propionate,iminothiolane, bifunctional derivatives of imidoesters such as dimethyladipimidate HCl, active esters such as disuccinimidyl suberate,aldehydes such as glutaraldehyde, bis-azido compounds such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates such as toluene2,6-diisocyanate, and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Carbon-14-labeled1-isothiocyanobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is a chelating agent suitable for conjugation of radionuclideto the antibody.

The cytotoxin component of the immunoconjugate can be a chemotherapeuticagent, a toxin such as an enzymatically active toxin of bacterial,fungal, plant or animal origin such as urease, or fragments thereof, ora small molecule toxin, or a radioactive isotope such as 212Bi, 131I,131In, 111In, 90Y, and 186Re, or any other agent that acts to inhibitthe growth or proliferation of a senescent cell.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates include maytansinoids including DM-1 and DM-4,adriamycin, doxorubicin, epirubicin, 5-fluoroouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g. paclitaxel, and docetaxel, taxotere, methotrexate,cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosamide,mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin,teniposide, daunomycin, carminomycin, aminopterin, dactinomycin,mitomycins, esperamicins, 5-FU, 6-thioguanine, 6-mercaptopurine,actinomycin D, VP-16, chlorambucil, melphalan, and other relatednitrogen mustards. Also included are hormonal agents that act toregulate or inhibit hormone action on tumors such as tamoxifen andonapristone. Toxins and fragments thereof which can be used includediphtheria A chain, nonbonding active fragments of diphtheria toxin,cholera toxin, botulinus toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolacaAmericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria, officinalis inhibitor, gelonin,saporin, mitogellin, restrictocin, phenomycin, enomycin, and thetricothcenes. Small molecule toxins include, for example,calicheamicins, maytansinoids including DM-1 and DM-4, palytoxin andCC1065.

In an embodiment, the binding agent, optionally an antibody and/orbinding fragment thereof, optionally conjugated to a toxin or a label orthe nucleic acid is comprised in a composition. In an embodiment thecomposition comprises a diluent such as a saline solution for examplephosphate buffered saline solution (0.05-1.0%).

The present invention also provides, for therapeutic use, a vaccinecomprising any immunogenic form of an epitope that is unique to amisfolded form of a senescent cell surface protein, to treat subjectspresenting with disease that is associated with senescent cellspresenting that misfolded form of the protein. Such carriers will benontoxic to recipients at the dosages and concentrations employed.Ordinarily, the preparation of such compositions entails combining theparticular vaccine antigen with saline, buffers, antioxidants such asascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins, amino acids, carbohydrates including glucose ordextrins, or chelating agents such as EDTA, glutathione and otherstabilizers and excipients. Such compositions may be in suspension,emulsion or lyophilized form and will be pharmaceutically acceptable;i.e., suitably prepared and approved for use in the desired application.Preferred peptide vaccine compositions also comprise an adjuvant. DNAadjuvants are preferred for human use. The peptides may be formulated asfusions with other immunogenic peptides from the same or a differentpathologic entity. Peptides may be synthesized as fusions of theepitopes identified herein with one or more T-helper epitope such asPADRE (SEQ ID NO: 118) or certain known tetanus peptides. Spacerpeptides also may comprise part of these fusions. Materials havingadjuvant activity are well known. Currently, however, Alum (Al(OH)₃),and similar aluminium gels are the only adjuvants licensed for humanuse. Other materials are also known to have adjuvant activity, and theseinclude: Freund's complete adjuvant, a water-in-mineral-oil emulsionwhich contains killed, dried mycobacteria in the oil phase; Freund'sincomplete adjuvant, a weaker formulation without the mycobacteria;saponin, a membrane active glucoside extracted from the tree Quilliasaponaria; nonionic block copolymer surfactants, non metabolisedsynthetic molecules which tend to bind proteins to cell surfaces;ISCOMS, lipid micelles incorporating Quil A (saponin) which mimic, inphysical terms, infectious particles; and muramyl dipeptide, a leukocytestimulatory molecule that is one of the active components of killedmycobacteria.

It will be appreciated that the vaccines noted above may comprise,instead of the epitope designated above, a variant thereof thatincorporates 1, 2 or 3, amino acid additions, substitutions ordeletions. Particularly the epitope may be a variant that has beentruncated or extended to consist of 6, 7, or 8 amino acids, preferably 7amino acids, and that incorporates up to 2, usually 1, amino acidsubstitution, for instance in which an amino acid is replaced by anoxidized form thereof, or an enantiomeric alternative thereof. It isapparent to those skilled in the art that substitution of certain aminoacids in these epitopes will not affect immunoreactivity toward theepitopes. For example, substitution of leucine by isoleucine or valineand all combinations thereof is unlikely to alter the sensitivity of anantibody raised against this epitope. Thus all epitopes capable ofgenerating antibodies reactive to the epitopes listed above for thepurpose of selectively identifying misfolded specific protein areaspects of this invention.

It is occasionally desirable to derivatize amino acids present in theepitopes to obtain a more robust immune response or more selectivereactivity toward the misfolded form. For example, a cysteine that onmisfolding of its host protein may become oxidatively derivatized tocysteine sulfinic acid or cysteine sulfonic acid (cysteic acid). Thusantibodies against a free peptide containing, for example, a cysteicacid residue in place of cysteine are potentially more specific to themisfolded form of the protein. In general, candidate epitopes identifiedaccording to the methods described herein and containing derivatives oftheir constituent amino acids are an aspect of the present invention.

For epitopes containing proline, it may be desirable to prepare antigenpeptides containing proline analogues that are fixed in the cis- ortrans-configuration. Such analogues have been described previously(Scheraga et al, J Am Chem Soc 121 (49), 11558 (1999); Wang et al, J OrgChem 68 (6), 2343 (2002)). Unlike the other amino acids, for which thereis a prohibitively large energy difference between the cis- andtrans-amide bond stereoisomers, proline in unstructured peptides is ableto interconvert between a cis- and trans-geometry on a relatively rapidtime scale. When a proline is incorporated into the folded protein,steric interactions lock it into only one of the two possibleconformers, but on unfolding it is free to racemerize. By raisingantibodies against peptides incorporating a proline analogue with theopposite stereochemistry to that present in the native structure, theselectivity of the antibody for the unfolded state is much increased.Thus epitope peptides predicted by the method and incorporating cis- ortrans-analogues of proline are an aspect of this invention.

In embodiments of the present invention, vaccines useful in thetreatment of disease include the following:

a) a vaccine that incorporates an immunogen comprising the epitopedesignated by SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14and 15, and particularly 1, 2, 5 and 9, for the depletion of senescentcells presenting misfolded PrP protein;b) a vaccine that incorporates an immunogen comprising the epitopedesignated by SEQ ID No. 82, 83, 84, 85 and 86 for the depletion ofsenescent cells presenting misfolded CD38;c) a vaccine that incorporates an immunogen comprising the epitopedesignated by SEQ ID No. 47, 48, 49, 50 or 51 for the depletion ofsenescent cells presenting misfolded CD44;d) a vaccine that incorporates an immunogen comprising the epitopedesignated by SEQ ID No. 52, 53, 54, 55 or 56 for the depletion ofsenescent cells presenting misfolded Fas ligand;e) a vaccine that incorporates an immunogen comprising the epitopedesignated by SEQ ID No. 57, 58, 59, 60, or 61 for the depletion ofsenescent cells presenting misfolded NOTCH1;f) a vaccine that incorporates an immunogen comprising the epitopedesignated by SEQ ID No. 62, 63, 64, 65, and 66, and preferably SEQ IDNos. 62 and 65, for the depletion of senescent cells presentingmisfolded Fas receptor; andg) a vaccine that incorporates an immunogen comprising the epitopedesignated by SEQ ID No. 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80 and 81 for the depletion of senescent cells presenting misfoldedEGFR.

In therapeutic use, the antibodies and corresponding fragments andconjugates that bind selectively to the senescent cell marker, andvaccines that elicit such antibodies, can be used to treat subjectspresenting with or at risk for a disease associated with cell senescenceincluding particularly the so-called aging diseases and conditions,including degenerative disease. The terms “treat”, “treatment,”“treating”, “therapeutic use,” or “treatment regimen” encompassprophylactic, palliative, and therapeutic modalities of administrationof the compositions of the present invention, and include any and alluses of the present products that remedy a disease state, condition,symptom, sign, or disorder caused or associated with, either directly orindirectly, a senescent cell presenting a misfolded form of a protein,including an inflammation-based pathology, infectious disease, allergicresponse, hyperimmune response, or other symptom to be treated, or whichprevents, hinders, retards, or reverses the progression of symptoms,signs, conditions, or disorders associated therewith.

The term “subject” generally refers to mammals and other animalsincluding humans and other primates, companion animals, zoo, and farmanimals, including, but not limited to, cats, dogs, rodents, rats, mice,hamsters, rabbits, horses, cows, sheep, pigs, elk or other ungulates,goats, poultry, etc. A subject includes one who is to be tested, or hasbeen tested for prediction, assessment or diagnosis of a disease ordisorder associated with cell senescence. The subject may have beenpreviously assessed or diagnosed using other methods, such as those incurrent clinical practice, or may be selected as part of a generalpopulation (a control subject). A subject may be a transgenic animal,e.g. a rodent, such as a mouse, that produces a target proteinespecially in misfolded form, or is lacking expression thereof (e.g. a‘knock-out’ mouse). For example, the subject may a transgenic mouseoverexpressing a normal form of the target protein or may be a wild-typemouse or hamster that has been infected with a misfolded form of thetarget protein.

For treatment, the binding agent, such as the immunogen used for activeimmunization and the antibody used for passive immunization are used in“effective amounts”. These are amounts useful, in a treatment regimen,to reduce the effect of the senescent cells that present the misfoldedprotein target. It will be apparent that the present invention isapplicable to a wide variety of diseases, and that the particular amountand treatment regimen effective to reduce the effect of the endogenousprotein will vary with each disease or condition, in accordance withestablished clinical practice.

In addition to such vaccines, the present invention provides for thetherapeutic use of binding agents such as antibodies in the treatment ofsubjects presenting with the conditions noted above, includingconditions/diseases related by the presence of the given misfoldedprotein. For treatment, antibody that binds selectively to the targetepitope is administered as a pharmaceutical composition, comprising theantibody and a pharmaceutically acceptable carrier, in dosage form.

Also provided in another aspect, is a pharmaceutical compositioncomprising a binding agent that is selectively toxic to senescent cellsin an amount effective to reduce the number of such cells for use indepleting senescent cells endogenous to a subject, wherein the bindingagent binds selectively to a senescent cell surface protein having amisfolded conformation.

Yet a further aspect includes a pharmaceutical composition comprising abinding agent that selectively binds selectively to a senescent cellsurface protein having a misfolded conformation.

For antibodies, fragments and conjugates, the dosage form is optionallya liquid dosage form. Antibody solutions can be prepared in watersuitably mixed with a surfactant such as hydroxypropylcellulose or anemulsifier such as polysorbate. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, DMSO and mixtures thereof with orwithout alcohol, and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms. Conventional procedures and ingredients for theselection and preparation of suitable formulations are described, forexample, in Remington's Pharmaceutical Sciences (2003-20th edition) andin The United States Pharmacopeia: The National Formulary (USP 24 NF19)published in 1999. Formulations optionally contain excipients including,but not limited to, a buffering agents, an anti-oxidant, a stabilizer, acarrier, a diluent, and an agent for pH adjustment. The pharmaceuticalforms suitable for injectable use include sterile aqueous solutions ordispersion and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl, or benzyl alcohol; alkyl parabens such asmethyl or propyl paraben; catechol; resorcinol; cyclohexanol;3-pentanol; and m-cresol); low molecular weight (less than about 10residues) polypeptides; proteins such as serum, albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginineor lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN, PLURONICS orpolyethylene glycol (PEG).

In treatment, the dose of antibody optionally ranges from about 0.0001mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.15mg/kg to about 3 mg/kg, 0.5 mg/kg to about 2 mg/kg and about 1 mg/kg toabout 2 mg/kg of the subject's body weight. In other embodiments thedose ranges from about 100 mg/kg to about 5 g/kg, about 500 mg/kg toabout 2 mg/kg and about 750 mg/kg to about 1.5 g/kg of the subject'sbody weight.

For example, depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of antibody or conjugate is acandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage is in the range from about 1 μg/kg to100 mg/kg or more, depending on the factors mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. However, other dosage regimens may be useful.Unit doses can be in the range, for instance of about 5 mg to 500 mg,such as 50 mg, 100 mg, 150 mg, 200 mg, 250 mg and 300 mg. The progressof therapy is monitored by conventional techniques and assays.

Therapeutic use of an antibody according to the present inventionentails antibody administration, by injection or infusion, to subjectspresenting with a disease in which cells or fluids present the epitopetargeted by the antibody, i.e., in which the misfolded target protein ispresent. Subjects that would benefit from treatment can be identified bytheir clinical features, together with examination of tissue samples orbodily fluids to identify cells that present the epitope targeted by theantibody, as discussed infra.

In embodiments of the present invention, antibody compositions useful inthe treatment of disease include compositions that incorporate thefollowing:

a) an antibody that binds selectively to the epitope designated by SEQID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, andparticularly 1, 2, 5 and 9, for the depletion of senescent cellspresenting misfolded PrP protein;b) an antibody that binds selectively to the epitope designated by SEQID No. 82, 83, 84, 85 and 86 for the depletion of senescent cellspresenting misfolded CD38;c) an antibody that binds selectively to the epitope designated by SEQID No. 47, 48, 49, 50 or 51 for the depletion of senescent cellspresenting misfolded CD44;d) an antibody that binds selectively to the epitope designated by SEQID No. 52, 53, 54, 55 or 56 for the depletion of senescent cellspresenting misfolded Fas ligand;e) an antibody that binds selectively to the epitope designated by SEQID No. 57, 58, 59, 60, or 61 for the depletion of senescent cellspresenting misfolded NOTCH1;f) an antibody that binds selectively to the epitope designated by SEQID No. 62, 63, 64, 65, and 66, and preferably SEQ ID Nos. 62 and 65, forthe depletion of senescent cells presenting misfolded FasR; andg) an antibody that binds selectively to the epitope designated by SEQID No. 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 and 81 forthe depletion of senescent cells presenting misfolded EGFR.

For treatment with a vaccine, subjects are immunized on a schedule thatcan vary from once a day, to once a week, to once a month, to once ayear, to once a decade. A typical regimen includes an immunizationfollowed by booster injections at 6 weekly intervals. Another regimenconsists of immunization followed by booster injections 1, 2 and 12months later. Alternatively, booster injections will vary depending onthe immune response and the physiological condition of the subject. Forimmunization, the epitope-containing immunogen can be administered in adose that ranges from about 0.0001 microgram to 10 grams, about 0.01microgram to about 1 gram, about 1 microgram to about 1 mg, and about100 to 250 micrograms per treatment. In one embodiment the timing ofadministering treatment is at one or more of the following: 0 months, 2months, 6 months, 9 months, and/or 12 months. In one regimen, the dosingis at 2, 6, 9, and 12 months following the first immunization. Inanother regimen, the dosing is at 2 and 4 weeks following the firstimmunization, and then monthly afterwards. In an alternative regimen,the dosing varies depending on the physiological condition of thesubject and/or the response to the subject to prior immunizations. Theroute of administration optionally includes, but is not limited to,intramuscular and intraperitoneal injections. In one embodiment thecomposition is injected into the deltoid muscle.

The vaccine composition itself can further comprise adjuvants. Adjuvantsfor parenteral immunization include aluminum compounds (such as aluminumhydroxide, aluminum phosphate, and aluminum hydroxy phosphate). Theantigen can be precipitated with, or adsorbed onto, the aluminumcompound according to standard protocols. Other adjuvants such as RIBI(ImmunoChem, Hamilton, Mont.) can also be used in parenteraladministration.

The active ingredients to be used for in vivo administration will besterile. This is readily accomplished by filtration through sterilefiltration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release include semipermeable matrices of solid hydrophobicpolymers containing the antibody, which matrices are in the form ofshapes articles, e.g., films or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly (2-hydroxyethyl-methacrylate), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers such as injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate, andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Diagnostically useful compositions comprising the antibody willincorporate a carrier suitable for diagnostic purposes, such as asolution of saline or buffered saline including phosphate bufferedsaline, together with any desired stabilizers or preservatives. Ofcourse, the composition can be provided in a lyophilized form to prolongstorage stability.

In diagnostic use, the binding agent is exploited to detect the presenceof senescent cells. The senescent cells to be detected may be in asample obtained from any source, including a living subject. A subjectincludes one who is to be tested, or has been tested for prediction,assessment or diagnosis of a disease or disorder associated with a givenmisfolded protein target. The subject may have been previously assessedor diagnosed using other methods, such as those in current clinicalpractice, or may be selected as part of a general population (a controlsubject). A subject may be a transgenic animal, e.g. a rodent, such as amouse, that produces a senescent cell presenting a target protein inmisfolded form. For example, the subject may be a transgenic mouseoverexpressing a normal form of the target protein or may be a wild-typemouse or other rodent that has been infected with a misfolded form ofthe target protein.

To assist with the identification of subjects who are candidates fortreatment with the antibody or vaccine compositions of the invention,the present invention further provides for the detection of an epitopeby in vitro or in vivo diagnostic methods.

To detect the presence of a misfolded protein in any given sample, thepresent invention provides a detection method in which a samplesuspected to contain the misfolded protein is treated with an antibodyor binding fragment that binds selectively to an epitope presenteduniquely by the misfolded protein relative to the natively folded formof that protein; and determining whether an antigen:antibody complex hasformed, the formation thereof being indicative of the presence in thesample of a misfolded form of said protein. In one embodiment, theepitope is one that has been identified by applying an epitopeprediction method. In another embodiment, the epitope is one that is, oris comprised within, a peptide having any one of SEQ ID Nos.1-36 and41-86.

In a related embodiment, the labeled antibodies of the invention, orlabeled form of a binding fragment thereof, can be used in vivo to imagethe presence of the misfolded protein to which the antibody binds. Tothis end, the present invention provides an antibody or fragment in aform coupled to an agent useful for in vivo imaging, such as isotopes oftechnetium, gadolinium, and the like.

In situ detection of the binding to cancer cells bearing misfolded PrPcan also be performed using the present antibody or fragment, byimmunofluorescence or immunoelectron microscopy. For this purpose, ahistological specimen is removed from the patient, and a labeled form ofthe present antibody is applied to it, preferably by overlaying theantibody on a biological sample, in keeping with standardimmunohistochemistry techniques. This procedure also allows fordistribution of the PrP antigen to be examined within biopsied tumourtissue, to reveal only those sites at which PrP is presented inmisfolded form. It will be apparent for those skilled in the art that awide variety of histological methods are readily available for in situdetection.

More particularly, antibodies or binding fragments of the presentinvention may be used to monitor the presence or absence of antibodyreactivity in a biological sample, e.g., a tissue biopsy from brain,skin, liver, heart, kidney, pancreas, bowel, spleen, muscle, fat, skin,ovary and the like, from a cell, or from fluid such as cerebrospinalfluid, blood including plasma, urine, seminal fluid, and the like, usingstandard detection assays. Immunological assays may involve directdetection, and are particularly suited for screening large amounts ofsamples for the presence of senescent cells that present misfoldedsurface protein. For example, antibodies may be used in any standardimmunoassay format (e.g., ELISA, Western blot, immunoprecipitation, flowcytometry or RIA assay) to measure complex formation. Any appropriatelabel which may be directly or indirectly visualized may be utilized inthese detection assays including, without limitation, any radioactive,fluorescent, chromogenic (e.g., alkaline phosphatase or horseradishperoxidase), or chemiluminescent label, or hapten (for example,digoxigenin or biotin) which may be visualized using a labeled,hapten-specific antibody or other binding partner (e.g., avidin).Exemplary immunoassays are described, e.g., in Ausubel et al., supra,Harlow and Lane, Antibodies: A Laboratory Approach, Cold Spring HarborLaboratory, New York (1988), and Moynagh and Schimmel, Nature 400:105,1999. For example, using the antibodies described herein, misfolded PrPis readily detected at the cell surface using standard flow cytometrymethods. Samples found to contain labeled complex compared toappropriate control samples are taken as indicating the presence ofmisfolded PrP, and are thus indicative of a disease state amenable totreatment with the present antibodies.

Senescent cell screening results that are obtained with misfoldedprotein binding agents can be confirmed using any other test appropriatefor senescent cell detection. It is known, for instance that misfoldedPrP is detectable not only on senescent cells, but also on certaincancer cells particularly including ovarian cancer cells. In certaininstances, therefore, the present senescent cell detection method shouldbe accompanied by a test that is confirmatory for senescent cells. Inembodiments, the confirmatory test can be the established assay forB-galactosidase activity. It has been shown that senescent cells willuse B-galactosidase as a substrate when cultured at mildly acidic pH,such as pH 6.0. As described by Dimri et al in U.S. Pat. No. 5,795,728,incorporated herein by reference, senescent cells can be identified byculturing cells in the presence of a B-galactosidase substrate, such asX-gal, and at pH 6.0. The cultured cells or tissue are then fixed in asolution such as 2% formaldehyde, 0.2% glutaraldehyde and PBS. Theappearance of reaction products, revealed by staining, indicates thatthe cultured cell is senescent. Alternatively, any cell or tissue can beexamined for senescence by assaying for the presence of INK4a/ARFexpression, as described by Sharpless et al in U.S. Pat. No. 8,158,347,also incorporated herein by reference. An elevation in this expressionproduct indicates the tested cell is senescent. Of course, other methodsare useful to identify senescent cells, including measurement of theincorporation of labeled DNA precursors such as 3H-thymidine and BrdU ormeasurement of cell markers that are expressed only in proliferatingcells, such as PCNA or MTT. Senescent cells will test negative for thesemarkers.

Such assays can also be performed prior to administering a binding agentor immunogen described herein and/or to identify subjects to be treated.In an embodiment, the method comprises detecting senescent cells in asample containing cells to be screened according to a method describedherein, for example using a binding agent, B-galactosidease assay etc orcombinations thereof; and administering to the subject a binding agentthat is selectively toxic to senescent cells in an amount effective toreduce the number of such cells, wherein the biding agent bindsselectively to a senescent cell surface protein having a misfoldedconformation, relative to said protein in a natively foldedconformation. In an embodiment, the method comprises detecting senescentcells in a sample containing cells to be screened according to a methodmethod described herein, for example using a binding agent,B-galactosidase assay etc or combinations thereof; administering to thesubject a binding agent that binds selectively to a senescent cellsurface protein having a misfolded conformation, for example to treat anage-related disease.

To detect the presence of a senescent cell in a given sample, thepresent invention provides a detection method in which a samplesuspected to contain the senescent cell is treated with a binding agent,e.g., an antibody or binding fragment, that binds selectively to anepitope presented uniquely by the misfolded protein relative to thenatively folded form of that protein; and determining whether anantigen:antibody complex has formed, the formation thereof beingindicative of the presence in the sample of a senescent cell. In oneembodiment, the epitope is one that is, or is comprised within, apeptide herein described.

When applied in vitro, the detection method entails analysis of acell-containing sample of body fluid or tissue or organ from a subject,usually a subject suspected of having endogenous misfolded targetprotein. For example, the biological sample may a body fluid such ascerebrospinal fluid, blood, plasma, lymph fluid, serum, urine or saliva.A tissue or organ sample, such as that obtained from a solid orsemi-solid tissue or organ, may be digested, extracted or otherwiserendered to a liquid form—examples of such tissues or organs includecultured cells, blood cells, brain, neurological tissue, skin, liver,heart, kidney, pancreas, islets of Langerhans, bone marrow, blood, bloodvessels, heart valve, lung, intestine, bowel, spleen, bladder, penis,face, hand, bone, muscle, fat, cornea or the like, including cancerousforms thereof. A biological sample or samples may be taken from asubject at any appropriate time, including before the subject isdiagnosed with, or suspected of having a protein misfolding associateddisease or disorder, during a therapeutic regimen for the treatment oramelioration of symptoms of that disease or disorder, after death of thesubject (regardless of the cause, or suspected cause). Alternately, abiological sample may include donated body fluid or tissue, such asblood, plasma or platelets when in care of a centralized blood supplyorganization or institution.

The presence in the sample of a senescent cell presenting a misfoldedtarget protein is confirmed if the antibody forms a detectableantigen:antibody complex. The formation of such complex can bedetermined using a wide variety of protocols that include ELISA, RIA,flow cytometry, Western blots, immunohistochemistry and the like. Toreveal the complex and hence the presence of the epitope in the sample,the antibody desirably is provided as a labeled antibody by conjugationor coupling to an agent that is detectable either visually or with theaid of instrumentation. The agent, or label, is capable of producing,either directly or indirectly, a detectable signal. For example, thelabel may be radio-opaque or a radioisotope, such as 3H, 14C, 32P, 35S,123I, 125I, 131I; a fluorescent (fluorophore) or chemiluminescent(chromophore) compound, such as fluorescein isothiocyanate, rhodamine orluciferin; an enzyme, such as alkaline phosphatase, beta-galactosidaseor horseradish peroxidase; an imaging agent; or a metal ion.Alternatively, the epitope can be revealed using a labeled secondaryreagent that binds to the epitope antibody, such as a labeled antibodythat binds the epitope antibody, to reveal presence of the epitopeindirectly. The presence of an antibody:antigen complex may be detectedby indirect means that do not require the two agents to be in solution.For instance, the complex is detectable indirectly using flow cytometry,where the antibody binds to, and forms an antibody:antigen complex with,the epitope presented on the surface of an intact cell. The applicationof the antibodies for detection of cell-surface forms of the epitope isa very useful embodiment of the invention particularly for detection ofsenescent cells presenting such epitopes. Detection of such cells can beachieved using the well-established technique of flow cytometry. It willalso be appreciated that the antigen:antibody complex can also beidentified by non-antibody based methods, that include those which sortproteins based on size, charge and mobility, such as electrophoresis,chromatography, mass spectroscopy and the like.

In a related embodiment, the labeled antibodies of the invention, orlabeled form of a binding fragment thereof, can be used in vivo to imagethe presence of senescent cells that present the misfolded protein towhich the antibody binds. To this end, the present invention provides anantibody or fragment in a form coupled to an agent useful for in vivoimaging, such as isotopes of technetium, gadolinium, and the like. Inembodiments, the senescent cell detection method is conducted usingantibodies that bind selectively to misfolded human PrP. In specificembodiments, the antibodies are selected from 1A1, c-120 and YYR MAbs,and misfolded PrP binding fragments and conjugates thereof. In otherspecific embodiments, the antibodies are selected from 3d-19 and 3a-118,and misfolded FasR-binding antibody fragments and conjugates thereof.

In therapeutic use, the binding agents that bind selectively to amisfolded protein marker on the surface of senescent cells can be usedto treat patients or subjects presenting with or at risk for a diseaseassociated with senescent cells. For therapeutic use, passiveimmunotherapy can be adopted by administering binding agents that areantibodies or binding fragments thereof. In the alternative, activeimmunotherapy can be adopted using vaccines that elicit the productionof such antibodies.

For treatment, the active ingredient, such as the immunogen used foractive immunization and the antibody used for passive immunization areused in “effective amounts”. These are amounts useful, in a treatmentregimen, to reduce the effect of the endogenous senescent cells byeliminating or reducing the number of senescent cells endogenous to therecipient. It will be apparent that the present invention is applicableto a wide variety of diseases, and that the particular amount andtreatment regimen effective to reduce the effect of the endogenousprotein will vary with each disease, in accordance with establishedclinical practice for each disease.

An anti-senescence therapeutic according to the invention may beadministered with a pharmaceutically-acceptable diluent, carrier, orexcipient, in unit dosage form.

Any appropriate route of administration can be employed, for example,parenteral, intravenous, subcutaneous, intramuscular, intracranial,intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, or oraladministration.

For the treatment of subjects presenting with senescent cells positivefor misfolded target protein, the appropriate dosage of agent, e.g., anantibody, fragment or conjugate, will depend on the type of disease tobe treated, as defined above, the severity and course of the disease,whether the agent is administered for preventative or therapeuticpurposes, previous therapy, the patients clinical history and responseto the agent, and the discretion of the attending physician. The agentis suitably administered to the patient at one time or over a series oftreatments, in accordance with dosing regimens discussed above.

Indications

It is anticipated that depletion of endogenous senescent cells will havea therapeutic effect and benefit on a variety of aging diseases andconditions, including particularly the degenerative disorders thataccompany aging. More particularly, senescent cell depletion is expectedto provide improvements in 1) reducing the rate at which adipose tissueis lost, 2) reducing the rate at which muscle fibre diameter is reduced,and 3) reducing the rate at which skin tone deteriorates over time.These effects are likely to be seen more dramatically in agedrecipients, i.e. those at an age greater than 50 years, especially thoseaged greater than 60 years or more, such as 65 years, 70 years and 75years and greater. Also, candidate recipients include those whoselifestyle imposes age-accelerating effects, including tobacco smokersand users, alcohol and narcotic drug abusers, skin tanning enthusiasts,and the like. These are therefore preferred recipients of the presenttreatment method.

Particular conditions and diseases that can be treated by the presentmethod include sarcopenia. Sarcopenia is characterized first by a muscleatrophy (a decrease in the size of the muscle), along with a reductionin muscle tissue “quality,” caused by such factors as replacement ofmuscle fibres with fat, an increase in fibrosis, changes in musclemetabolism, oxidative stress, and degeneration of the neuromuscularjunction. Combined, these changes lead to progressive loss of musclefunction and frailty.

Other conditions that can be treated by the present method includecataracts, and so-called “signs of aging” such as wrinkling anddiscolouration of the skin, and overall dermal tone. Treatment by thepresent method is expected to reduce the rate at which fat and musclethat support skin tone are reduced, so that skin wrinkling also isreduced, delayed or eliminated. As well treatment is expected to have abenefit on the rate at which cataracts form in the eye.

It has been demonstrated that senescent cells create a local environmentthat supports tumorigenesis in neighbouring cells. Treatment by thepresent method can accordingly be useful to treat cancer in varioustissues, including cancers of the lung, prostate, skin, breast, and thelike. Such treatment is expected to result in a decrease in the rate oftumour formation directly, and thus indirectly also on the number, sizeand distribution of responsive cancer cells and tumours.

In specific embodiments of the present invention, antibody compositionsuseful in the treatment of disease include compositions that incorporatethe following:

a) an antibody that binds selectively to an epitope presented bypeptides designated by SEQ ID No. 1-37 and 47-86 for the treatment ofsubjects afflicted with sarcopenia; andb) an antibody that binds selectively to an epitope presented bypeptides designated by SEQ ID No. 1-37 and 47-86 for the treatment ofaged subjects presenting with signs of skin aging such as wrinkling,discolouration and the like; andc) an antibody that binds selectively to an epitope presented bypeptides designated by SEQ ID No. 1-37 and 47-86 for the treatment ofaged subjects presenting with tumours.

The invention also includes articles of manufacture as well as kits thatcomprise components useful to perform the diagnostic and therapeuticmethods of the present invention. The articles of manufacture comprisepackaging material and a composition comprising an antibody or antiserathat binds selectively to senescent cells that present an epitope uniqueto a misfolded form of protein, relative to the natively folded form ofthat protein. The composition includes a physiologically orpharmaceutically acceptable excipient, and the packaging material mayinclude a label which indicates the active ingredients of thecomposition (e.g. the antisera or antibody). The label may furtherinclude an intended use of the composition, for example as a diagnosticreagent to be used with kits as set out herein.

Also provided is an article of manufacture, comprising packagingmaterial and a composition comprising a peptide, or one or morepeptides, as provided herein. The composition may include aphysiologically or pharmaceutically acceptable excipient, and thepackaging material may include a label which indicates the activeingredients of the composition (e.g. the peptide). The label may furtherinclude an intended use of the composition, for example as a therapeuticor prophylactic reagent, or as a composition to induce an immuneresponse in a subject for the purpose of producing antisera orantibodies specific to senescent cell target protein, to be used withkits as set out herein.

In a further embodiment, there is provided a kit comprising acomposition comprising one or more peptides as provided herein, alongwith instructions for use of the compound or composition for theproduction or screening of antibodies for identification of senescentcells. The kit may be useful for production and/or identification ofsenescent cell-specific antibodies or antisera, and the instructions mayinclude, for example, dose concentrations, dose intervals, preferredadministration methods, methods for immunological screening or testing,or the like.

In another embodiment, a kit for the preparation of a medicament,comprising a composition comprising one or more peptides as providedherein, along with instructions for its use is provided. Theinstructions may comprise a series of steps for the preparation of themedicament, the medicament being useful for inducing a therapeutic orprophylactic immune response in a subject to whom it is administered.The kit may further comprise instructions for use of the medicament intreatment for treatment, prevention or amelioration of one or moresymptoms of a disease or disorder associated with protein misfolding onsenescent cells, or in which protein misfolding is implicated, andinclude, for example, dose concentrations, dose intervals, preferredadministration methods or the like.

In another embodiment, a kit for diagnosing a disease or disorderassociated with protein misfolding is provided. The kit comprises one ormore misfolded protein-selective antibodies or antisera as describedherein, along with instructions for its use. The antibody may further becoupled to a detection reagent. Examples of detection reagents includesecondary antibodies, such as an anti-mouse antibody, an anti-rabbitantibody or the like. Such secondary antibodies may be coupled with anenzyme that, when provided with a suitable substrate, provides adetectable colorimetric or chemiluminescent reaction. The kit mayfurther comprise reagents for performing the detection reaction,including enzymes such as proteinase K, blocking buffers, homogenizationbuffers, extraction buffers, dilution buffers or the like. The kit mayfurther comprise reagents useful to perform the confirmatoryB-galactosidase test for senescence, as discussed above.

In another embodiment, a kit for detecting the presence of senescentcells in a biological sample is provided. The kit comprises one or moreantibodies or antisera that specifically bind the misfolded proteinpresented by the senescent cells, along with instructions for its use.The antibody may further be coupled to a detection reagent. Examples ofdetection reagents include secondary antibodies, such as an anti-mouseantibody, an anti-rabbit antibody or the like. Such secondary antibodiesmay be coupled with an enzyme that, when provided with a suitablesubstrate, provides a detectable colorimetric or chemiluminescentreaction. The kit may further comprise reagents for performing thedetection reaction, including enzymes such as proteinase K, blockingbuffers, homogenization buffers, extraction buffers, dilution buffers orthe like. The kit may further comprise reagents useful to perform theconfirmatory B-galactosidase test for senescence, as discussed above.

Example 1 Antibodies to YYR Epitope of Misfolded Human PrP (See U.S.Pat. No. 7,041,807)

Epitopes uniquely exposed on misfolded, but not natively structured,PrPC reside in the peptides RYYRENMH and QVYYRPV (SEQ ID Nos 5 and 7respectively). Resident within each of these peptides is the 3-aminoacid epitope designated YYR noted above. Antibodies against the YYRepitope of PrP are described in the literature by Cashman (see U.S. Pat.No. 7,041,807 incorporated herein by reference). Some of that work isreproduced below. As noted in the results, the antibodies to YYR bindselectively to the disease-misfolded form of PrPC, and do not bind tothe natively folded form.

In order to develop an antibody to the YYR epitope presented bymisfolded PrPC and but not by natively folded PrPC, a peptide with theamino acid sequence Acetyl-Cys-Tyr-Tyr-Arg-NH2 (YYR) was synthesized,conjugated to KLH, and injected intramuscularly into rabbits using wellknown techniques. At the amino-terminus of the peptide, a cysteineresidue was added to allow conjugation of the peptide with the proteincarrier. The amino group of the peptide was blocked by acetylation, andthe carboxylic group of the peptide was blocked by amidation.

Peptides were synthesized using solid phase peptide synthesis methodseither manually or automated (MPS396 peptides synthesizer, AdvancedChemTech). Coupling of amino acid residues was accomplished using Fmocpeptide synthesis chemistry (Fields et al., 1990, IJPPR 35, 161).Syntheses were performed on Wang or on amide Rink resins, with full sidechain protection of amino acids. Since the alpha-NH2 groups of the aminoacids were protected with the Fmoc group, the following protectivegroups were chosen for the side groups of the trifunctional amino acids:

Cysteine: 5-triphenylmethyl (Trt)Arginine: 2,2,4,6,7-pentamethyldihydrobenzofuran-5 sulfonyl (Pbf)Tyrosine: tert.-butyl ether (tBu)\

BOP, PyBOP, or TBTU were used as activation agents, depending on thechemistry and difficulty of the coupling reaction. All chemicals werepurchased from Advanced Chem Tech, Bachem, and Calbiochem/NovaBiochem.Formation of each peptide bond between residues of the sequence wasensured by using a 3 to 6 fold excess of coupling reagents and byso-called double coupling; meaning that the coupling reaction wasrepeated for each amino acid added to the growing peptide chain.

After synthesis, the peptides were cleaved from the resin using theReagent K as a cleavage mixture: water (2.5%), TIS (2.5%), EDT (2.5%),TFA (92.5%). The peptides were then precipitated with cold diethylether. The precipitates were centrifuged, washed three times withdiethyl ether, dissolved in 20%-50% AcCN/water mixture, and lyophilized.Analysis of crude products was performed using analytical RP-HPLC andelectrospray MS.

The crude peptide was purified by Rp-HPLC (reverse phase highperformance liquid chromatography) on a Vydac C18 column, 2.5×25 cm,using a linear gradient of 10-50% acetonitrile in water, with 0.06% TFA(1%/min gradient, 10 ml/min flow rate), with monitoring by UV at 215 nmand 254 nm. Analytical HPLC was used to estimate the purity of thefractions. The final product was obtained as a lyophilized peptide withat least 95% purity estimated by analytical HPLC (Vydac C18, 0.46×25 cm,linear gradient 10-60% acetonitrile in water, 0.1% TFA, 1%/min, 1 mL/minflow rate, detection by UV absorption at 215 nm and 254 nm). The purepeptide was identified by molecular mass analysis using a SCIEX API IIImass spectrometer according to standard procedures.

The retention time of the peptide on RP-HPLC was 21.215 minutes. Thetheoretical molecular weight of the peptide was calculated to be 644.74;the actual molecular weight, through molecular mass analysis, was foundto be 646.5 (MW+H*).

Peptides were coupled to a carrier, in this case Keyhole limpethemocyanin (KLH). Other carriers useful for such coupling include,without limitation, albumin, or ovalbumin, 8map, or lysozyme. Couplingwas effected via a thioether linkage to the mercapto group of thecysteine. This type of linkage has the advantage that the peptide iscoupled in a defined way to a carrier protein.

Coupling to KLH was performed as follows. 10 mg of the peptide wasdissolved in 2 ml of phosphate buffered solution (PBS 1×). 1 ml of KLH(pierce products #77100) was added to the peptide solution and stirred(1 mole of peptide/50 amino acids). The KLH concentration was 10 mg/ml.20 ul of glutaraldehyde (25% aqueous solution) was added to thepeptide/carrier solution with constant stirring, incubated for 1 hour,after which a glycine stop solution was added. The peptide/carrierconjugate was separated from the peptide by dialysis against PBS

Polyclonal antibodies were prepared according to standard methods, andan immune response was enhanced with repeated booster injections, atintervals of 3 to 8 weeks. The success of the immunization was verifiedby determining the concentration of antibodies in a western blot orELISA or both. More specifically, to generate polyclonal antibodies tomisfolded PrPC (or PrPSc), the tripeptide YYR conjugated to KLH wasinjected into rabbits in accordance with a 164 day immunization regimen,after which the animals that had produced specific antibodies were bled.

In order to sample the serum prior to immunization, 10 ml of blood perrabbit was taken as a preimmune control. Primary immunizations werecarried out with Freund's complete adjuvant and subsequent boosts withincomplete Freund's adjuvant (IFA0 (1 ml per rabbit, 0.5 ml per thighmuscle). Each injection consisted of approximately 200 ug of thepurified peptide. At days 21, 42 and 70, a booster injection was givenwith IFA. At days 31, 42 and 80, 10 ml of blood was collected from thecentral ear artery for titer determination (6 ml/kg/rabbit). At day 80,the titer of the sera was checked, and 3 more injections were given(IFA) at 4 week intervals, followed by blood sampling 10 days later. 10days after the last boost, anesthetized rabbits were exsanguinated viacardiac puncture, and antisera were collected.

Goat polyclonal antibodies were generated according to standard methods.Three goats were immunized as follows. On day 1, all the goats receiveda primary immunization of 1 mg of YYR-KLH conjugates in completeFreund's adjuvant. Boosts were done by injection of 1 mg YYR-KLH inincomplete Freund's adjuvant for two of the three goats, whereas thethird goat received 1 mg YYR-8map conjugates in incomplete Freund'sadjuvant. Serum samples from each of the three bleeds were tested forreactivity by ELISA against YYR-BSA conjugates. From the third set ofbleeds, total IgG was purified by ammonium sulfate precipitation andYYR-reactive IgG was purified using a YYR affinity column. IgG fractionswere tested for reactivity to PrPSc as described herein. The exactimmunization schedule was as follows: Day 1, primary immunization; D 21,first boost immunization; Day 30, first bleed; Day 46, second boostimmunization; Day 53, second boost immunization; Day 60, second bleed;Day 76, third boost immunization; Day 83, third boost immunization; andDay 90, third bleed.

Alternatively, monoclonal antibodies may be prepared using the syntheticpeptides described herein and standard hybridoma technology (see, e.g.,Kohler et al., Nature 256, 1975; Kohler et al., Eur. J. Immunol. 6:511,1976; Kohler et al., Eur. J. Immunol. 6:292, 1976 Hammerling et al., InMonoclonal Antibodies and T Cell Hydridomas, Elsevier, NY, 1981; Ausubelet al., 1999, Current Protocols in Molecular Biology, WileyInterscience, New York,) Once produced, monoclonal antibodies are alsotested for specific PrP recognition by immunoprecipitation and westernblot analysis.

The generation of monoclonal antibodies was carried out as follows. Micewere immunized with baculovirus supernatant containing mouse PrP-APfusion protein in complete Freund's adjuvant, then boosted 2 weeks laterwith the same antigen in incomplete Freund's adjuvant. Two weeks afterthat immunization the mice were boosted with a mixture of PrP-APsupernatant plus 100 ug of KLH-CYYRRYYRYY (SEQ ID NO: 90 and 10 ug ofKLH-CKYEDRYYRE (SEQ ID NO: 91) conjugates. Splenocytes from these micewere fused to the FO murine B cell line (ATCC CRL-1646) to generatespecific hybridoma clones. Hybridoma supernatants were screened byELISA. There were no reactive supernatants to PrP-AP or to theCKYEDRYYRE (SEQ ID NO: 91) sequence, although there were clones reactiveto YYR-8map conjugates.

Total rabbit IgG was purified from serum using the Pharmacia protein AHiTrap column according to the manufacturer's recommendations. Briefly,a HiTrap column was equilibrated with 3 column volumes of start buffer(0.2M sodium phosphate buffer, pH7.0). Serum was applied, using asyringe through a luer adaptor, onto the column. The column wassubsequently washed with 5 ml of start buffer. Bound protein was elutedwith 0.1M glycine, pH 3.0, and collected in eppendorf tubes containing1M Tris pH 8.0 (50 ul/500 ul sample). Fractions were analyzed onSDS-PAGE.

Goat polyclonal antibodies were purified from serum samples as describedabove.

Mouse monoclonal antibodies were produced as ascites, and purified usinga protein A column kit (Pierce) according to the manufacturer'sinstructions. Briefly, a sample of ascites was diluted with bindingbuffer at a 1:1 final ratio. The sample was then added to the top of thecolumn, which had been previously equilibrated with binding buffer, andallowed to flow through the matrix. The pass-through material wascollected and the column washed with 5 volumes of binding buffer. Mildelution buffer was added to the column to release the bound IgG antibodyfrom the matrix. Other antibody isotypes were collected by switching tothe IgG elution buffer. All the antibodies were collected in 1 mlfractions, which were analyzed by BCA to determine total protein contentand SDS-PAGE electrophoresis to establish the degree of antibody purity.The fraction containing the most yield of IgG was desalted by passing itthrough a D-salt column (Pierce). The antibody fraction was allocatedand stored at −80 C. in PBS

Antibodies produced using the aforementioned procedures weresubsequently tested for high-affinity binding as follows.

Ten ul of brain extract was added to 950 ul of Immunoprecipitationbuffer (PBS 3% NP-40, 3% Tween-20) and incubated at 37° C. for 30 or 60minutes. For experiments evaluating the reactivity of PrP 27-30 with thebead conjugates, the incubation was preceded by addition of 50 ul of 1mg/ml proteinase K. Samples not treated with proteinase K were stillincubated at 37 D. for the appropriate time period. After theincubation, 60 ul of a 100 mM PMSF solution were added to both sets oftubes. On hundred ul of resuspended bead conjugates were then added tothe mixture, and incubated with rotation at room temperature for 2hours. The beads were washed 3 times with washing buffer (PBS 2% NP-402% Tween-20) and resuspended by vortex after each wash. After the lastwash, the beads were resuspended in 20 ul of 2× loading buffer (100 mMTris pH 6.8, 4% SDS, 0.015% bromphenol blue, 20% glycerol) and heated at95° C. for 3 minutes.

The PrPSc content of brain homogenates was determined by westernblotting according to standard methods. Protein samples were mixed with2× sample buffer at a ratio of 1:1 and boiled for 5 minutes at 100° C.SDS-PAGE analysis was performed according standard methods. Samples wereapplied to a pre-cast 15% acrylamide gels (Biorad) along withpre-stained molecular weight markers (Biorad). The gels were run at 100V until the bromophenol blue dye front reached the bottom of the gel.The separated protein was then transferred onto PVDF membranes at 100 Vor 1 hr. The membranes were washed as described above before incubationwith a goat anti-mouse IgG alkaline phosphatase conjugated secondaryantibody (1:5000 in TBST) for 1 hour at room temperature. After washing,signals were developed with the chemiluminescent substrate CDP-star, andexposed to x-ray films.

Spleen cell suspensions were prepared from Balb/c mice by passing thetissues through a wire mesh. The cells were washed once with coldDulbecco's PBS without Ca2+ or Mg2+ and viable cells were isolated byunderlayering of the cell suspension with Lympholyte (Cedarlane) andcentrifugation at 1300 g for 20 minutes. The cells were washed once withcold Dulbecco's PBS without CA2+ or Mg2+2.5% fetal bovine serum, and0.5×10⁶ cells were aliquoted per well in a round bottom 96 well plate.The cells were centrifuged and resuspended in 50 ul of antibody-FITCconjugates at 1/10 final concentration in Dulbecco's PBS without CA2+ orMg2+2.5% fetal bovine serum, for 15 minutes on ice. The cells were thenwashed twice with cold Dulbecco's PBS without Ca2+ or Mg2+2.5% fetalbovine serum and resuspended in the same medium containing 1 ug/ml ofpropidium iodide. The cells were analyzed on a Coulter Epics flowcytometer and were gated by size and granularity (forward and sidescatter) and viability (exclusion of propidium iodide fluorescence).

Fluoresceinated mAbs were made by using the Fluorotag kit (Sigma)following the manufacturer's instructions. Briefly, 0.5 mg of eachantibody was raised to pH 9 with concentrated bicarbonate buffer, andFITC stock solution was added to produce an FITC: antibody ratio of20:1. The vials were then incubated for 2 hours at room temperature.Labeled antibody was separated from free FITC by passing the mixtureover a Sephadex G-25M column. Conjugated antibodies were tested forsuccessful fluoresceination by measuring their FITC emissions at 35 nmusing an LJL Biosystems Analyst, and the antibodies were tested forretention of their binding activity with an ELISA against YYR-8mapconjugates.

To determine whether antibody pAbC2 was useful in specificallyrecognizing PrPSc from bovine brain extracts, compared to PrPC usingrecombinant PrP (rbPrP), an ELISA approach was used. Either pools ofPrPSc containing brain extracts or rbPrP was used to test thespecificity of pAbC2 for PrPSc. The wells of an Immunolon ELISA plate(Dynex) were coated overnight at 4° C. with the PC2 containing culturesupernatant in a TBS buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl,1 mM CaCl₂. For BSE-brain extract experiments, control wells were coatedwith a supernatant containing Mek-4; for rbPrP experiments, milk wasused as a control to determine the non-specific binding of the antibodyto the well. The coating of the ELISA plates with soluble PC2 wasconfirmed with an anti-PC2 monoclonal antibody. The wells were washedfour times using a SLT ‘Columbus’ microplate washer (Tecan) with TBScontaining 0.05% Tween 20, and blocked by filling the wells with 0.2%I-Block (Tropix) in TBST and incubating the plate at 37° C. for 1 hour.The plates were washed and the bovine brain homogenate (diluted to 1%w/v in TBS) or rbPrP was added to designated wells and incubated at RTfor 1 h. Wells were washed four times with TBST. pAbC2 was added toappropriate wells and incubated at RT or 1 hour, followed by a further45 minute incubation with 100 ul of an anti-rabbit or mouseIgG/horseradish peroxidase conjugate (1:5000) in TBST containing 1%non-fat milk. Wells were washed four times with TBST. Signals weredeveloped with TMB/H2O2 as a substrate for peroxidase. Reactions werestopped after 15 minutes by the addition of 100 ul of 2M phosphoricacid. Signals were monitored at 450 nm with reference at 620 nm using aSLT microplate reader. Specific positive signals were determined bycomparing PrP binding to PC2 with PrP binding to the negative control,Mek-4 or milk. Preimmune controls showed no binding.

It will thus be appreciated that various antibodies that recognize andbind selectively to the YYR epitope unique to misfolded PrPC can beobtained by raising antibodies against those epitopes in accordance withstandard practices well established for these purposes.

Example 2 Antibodies to YML Epitope of Misfolded Human PrP (See WO2010/099612)

As shown in Table 1, a region of PrPC beta strand 1 contains the epitopedesignated YML as shown by peptides LGGYML and GGYMLGS of SEQ ID Nos 1and 2, respectively. In the experiment that follows, the monoclonal IgMantibody 1A1 was raised specifically against the YML epitope using, asantigen/immunogen, a peptide having the sequence GGYMLGS (beta strandone with two flanking residues N and C terminal, SEQ ID No.2). This workis also described in WO 2010/099612, incorporated herein by reference.

General references: 263K hamster-adapted prions are described byKimberlin et al., 1978. RML mouse-adapted prions are described byChandler, R. L. (1961) (Lancet 1, 1378-1379). These may be used toinfect mice or hamsters, using methods known in the art, for examplethose of Bueler H et al., 1993 (Cell 73:1339-1347), Oldstone et al.,2002, or Meade-White et al., 2009. Bolton et al., 1987 describe methodsthat may be used for isolation and purification of scrapie agent.Carlson et al., 1986 (Cell, 46:503-511) describes methods that may beused for clinical diagnosis of scrapie in mice and hamsters. Varioustransgenic mice overexpressing, partially expressing or lackingexpression of PrP are described by Fischer et al., 1996 (EMBO J15:1255-1264) and Weissmann et al., 2003 (British Medical Bulletin66:43-60).

Brain tissues (normal and scrapie-infected mouse or hamster) wereprocessed and analyzed as modified from Fischer et al 2000 (Nature408:479-483). Briefly, 10% homogenates were made in PBS, 0.5%deoxycholate (Sigma), 0.5% NP-40. The total protein concentration in thehomogenate was determined by BCA assay (Pierce) and adjusted to 5 mg/mlwith homogenization buffer. For detection of PK-resistant material, 1.5μl of homogenate was incubated with, or without, 0.15 μg PK (Sigma) at37° C. for 60 minutes. Digestion was halted by addition of 20 mM PMSF(Sigma).

Antibodies were conjugated to magnetic beads and used forimmunoprecipitation experiments as modified from Paramithiotis, 2003.Briefly, 7×10⁸ magnetic beads (in 1 ml PBS) (Dynal; Lake Success, NewYork) were coupled according to the manufacturer's instructions to: 1A1,8B4, 4E4 or IgM isotype control. Conjugated beads were washed andblocked according to the manufacturer's recommendations, thenresuspended in 1 ml of PBS.

10 μL of antibody-coupled beads were incubated with 1 μL of 10% brainhomogenate in 6% detergent (3% Tween20 and 3% NP40 in PBS) for 3 hr atroom temperature. Magnet-captured immune complexes were washed 3 timeswith 4% detergent (2% Tween20 and 2% NP40 in PBS), boiled in 4% SDSwithout reducing agents, and resolved on 15% Trisglycine or 4-12%bis-tris acrylamide gels (Invitrogen).

Immunoblotting was performed as described (Paramithiotis et al., 2003).Proteins were transferred onto PVDF membranes (Invitrogen). Membraneswere blocked with 5% (w/v) dried non-fat milk. All incubations were donein TBST (25 mM Tris-HCl, 0.2 M NaCl, 0.5% Tween-20). Peroxidase activitywas detected by chemiluminescence; enhanced ECL (Amersham) or superWestDura (Pierce; Rockford, Ill.).

Immunoprecipitated samples were analyzed by Western blotting with6D11-biotin as the primary antibody (1:5000) and Strep-HRP as thesecondary antibody (1:5000). 8B4-bead acted as a positive control andwas able to immunoprecipitate PrP from all the brain homogenate samplesexcept the PrP Knocked Out mouse (K/O). Beads only, IgM-isotype-beadsand 4E4-beads acted as negative controls and as expected, no PrP wasimmunoprecipitated, except two very faint bands in the RML and 263Klanes as immunoprecipitated by the 4E4. 1A1, an IgM antibody that wasraised against the beta-1 strand of PrP, was able to immunoprecipitatescrapie prion proteins from both RML (mouse scrapie strain) and 263K(hamster scrapie strain). There is a faint band in the Tg20 lanepossibly due to a small content of misfolded PrP in this Prnpoverexpressing transgenic mouse brain. Data indicated that 1A1 was ableto recognize only the scrapie PrP, but not the wild type PrP in bothmouse and hamster brain.

Example 3 YML on the Surface of Tumors

The 1A1 antibody was tested for its ability to bind to both normal andtumor cells. As a control, the anti-PrPC antibody 6D11 was used toidentify the level of expression of PrP on each cell type. The abilityof antibodies to bind to cells was observed by flow cytometry usingHUVEC (human umbilical vein endothelial cells) as the normal cell type.Eight types of tumor cells were tested. Five of the cancer cells testedare immortalized cell lines from mice (B16—melanoma, NSC34—motorneuron/neuroblastoma hybrid) and humans (HL60—promylocytic leukemia,MO3.13—oligodendrocyte/muscle hybrid, SiHa—cervical carcinoma). Theremaining cancer cells tested are primary tumor cells that have beenpropagated by the Living Tumor Laboratory (LTL) at the British ColumbiaCancer Agency. Using proprietary technology, primary human tumors arepropagated under the kidney capsules of immunodeficient mice. Thisallows the original tumor architecture and phenotype to remainconsistent with the originally harvested tumor. The three tumors thathave been tested for binding to the 1A1 antibody are LTL-013 (largediffuse B-Cell lymphoma), LTL-257 (colorectal sarcoma) and LTL-323(melanoma).

By flow cytometry, the 1A1 YML monoclonal antibody shows minimal bindingto normal HUVEC cells compared to an isotype IgM control. 1A1 also showsno detectable binding to HL60 myeloid leukemia compared to IgM isotypecontrol. HUVECs and HL60 cells have moderate and high levels,respectively, of 6D11 immunoreactivity, indicating that YML exposure isnot a simple function of prion protein expression. The 1A1 antibody alsoshows detectable binding to seven other human and mouse tumor cell linesand LTL human tumors, all of which also display detectable cell surface6D11 prion protein immunoreactivity.

To determine if an anti-DSE antibody can modify tumor progression invivo, the 1A1 antibody was tested for its ability to modify growth of amurine melanoma tumor (B16) in female C57B1/6 mice. On day 0 of thestudy, 3×105 tumour cells were implanted subcutaneously into the flankof 12 mice. The mice were randomly assigned to two treatment groups.Group 1 was treated with PBS. Group 2 was treated with 1A1 antibody at10 mg/kg. Mice were treated on days −1, 2 and 5.

Tumour growth was monitored by measuring tumour dimensions with calipersbeginning on day 2. Tumour length and width measurements were obtainedand tumour volumes were calculated according to the equation L×W2/2 withthe length (mm) being the longer axis of the tumour. Mice weresacrificed once tumour burden was high, according to standard animalcare procedures. There is a significant difference in tumour growthbetween the two groups (paired t-test=0.012; Wilcoxin=0.007), indicatingthat a therapeutic effect of the 1A1 antibody has occurred.

Example 4 Antibodies to the Rigid Loop Epitope of Misfolded Human PrP

Peptides comprising the sequence MDEYSNQNN (SEQ ID No. 9) weresynthesized using standard methods and then coupled to carrier proteins.Prepared immunogens included both KLH-Cys-MEDYSNQNN andOVA-Cys-MDEYSNQNN. This work is described in the applicants co-pendingU.S. Ser. No. 61/658,569 filed Jun. 12, 2012 and incorporated herein byreference.

New Zealand white rabbits were immunized subcutaneously with 0.4 mgpeptide-KLH conjugates in complete Freund's adjuvant. After the initialimmunization, animals were boosted several times every 2-3 weeks. Therabbit with the best titer in immunoassay was intravenously boosted withpeptide antigen again, four days before the removal of the spleen. Thehybridoma fusion was performed using conventional PEG cell fusionmethodology. Splenocytes were harvested from the immunized rabbit andfused with rabbit plasmacytoma cells 240E-W2 (U.S. Pat. No. 5,675,063)using PEG4000 (Sigma Chemical, St. Louis, Mo.) and selected by HAT(hypoxanthine, aminopterin, and thymidine). At the end of selectionhybridoma supernatants were collected and evaluated in various assays.Selected hybridomas were subsequently subcloned by limited dilution toobtain monoclonal hybridomas.

The antibody heavy and light chain genes for monoclonal ab120 werecloned from the hybridoma cells. Total RNA was extracted andreverse-transcribed to cDNA using the Qiagen TurboCapture mRNA kits. DNAfragments for L chain and the variable region (VH) of H chain of rabbitIgG were amplified by PCR with rabbit H and L chain primers. The L chainfragment was cloned into pTT5 mammalian expression vector and the VHfragment fused in-frame to the constant region of H chain pTT5 Heavychain vector For each hybridoma clone, three plasmid DNA clones for Hand L chains were sequenced and expressed as recombinant RabMAb forcharacterization.

Plasmids encoding the IgG heavy and light chains of ab120 were isolatedfrom transformed E. coli using EndoFree® plasmid purification kit(Qiagen). Human HEK-293-6E cells were used for transient expression ofab120 antibody. The antibody plasmids were transfected into cells atlogarithmic growth phase using FreeStyle™ MAX Reagent 293 fectin(Invitrogen, Cat: 51-0031) and cultured in FreeStyle™ 293 ExpressionMedium (Invitrogen, Cat: 12338-18) according to manufacturer'sinstructions. The transfected cells were grown at 37° C. with 5% CO2 inan orbital shaker for 7 days. The antibody secreted into the culturemedium was collected by spinning at 7000 rpm for 15 minutes to removecell debris. The cleared culture supernatant was purified by protein Achromatography (HiTrap™ rProtein A FF, GE healthcare, CAT: 17-5080-01)under endotoxin free condition. Antibodies were eluted from the columnin citrate elution buffer (SIGMA, CAT: C2404-100G) and adjusted toneutral pH with sodium bicarbonate buffer. The antibody preparation wasconcentrated and exchanged into PBS buffer. The concentration of IgG andendotoxin level in the final antibody preparation were determined by OD280 nm quantitation and Tachypleus Amebocyte Lysate gel clot assay(Zhanjiang A&C Biological Ltd), respectively.

Monoclonal antibody ab120 was purified by protein A. Purified antibodywas filter-sterilized and stored at 4 C in PBS buffer (pH 7.4). Theprotein concentration was determined by UV absorption 280 nm) assay andPBS buffer was used a blank buffer. The final concentration is the meansfrom triplicate readings, and was given a QC requirement of >2 mg/ml.

To measure protein purity, SDS-PAGE was performed with Bio-Rad minielectrophoresis system according to the manufacturer's instructions. Thegel was then stained with Coomassie brilliant blue. The resolving gelwas 12% acrylamide and the stacking gel was 4% acrylamide, with sampleloading at 4 ug/lane. The assayed sample showed 2 bands (Heavy chain andLight chain) in reduced SDS-PAGE, and one band (whole IgG molecule) innon-reduced SDS-PAGE.

Endotoxin level was also assessed by the Gel Clot Tachypleus AmeboycteLysate (TAL) kit using endotoxin standards and endotoxin-free water.Results indicated an endotoxin level of <1 EU/ml protein.

Thus in a preferred embodiment, the antibody is provided as apreparation that exhibits (a) <about 1 EU/ml protein, (b) aconcentration of greater than about 2 mg/ml, (c) and migration as asingle protein band when measured by non-reducing SDS-PAGE at a loadingdose of 4 ug/lane and detected at 280 nm.

Maxisorp 96-well plates were coated overnight at 2-8° C. with 100ng/well of BSA-peptide in PBS. After blocking with PBST/casein, primaryantibodies were added and incubated for 1 hour at room temperature.Rabbit antibodies were detected using goat anti-rabbit IgG-HRP and TMBsubstrate. After stopping the reaction with 0.25M sulfuric acid,absorbance was measured at 450 nm.

Recombinant PrP (Alicon) was mixed with LDS sample buffer (LifeTechnologies) and sample reducing agent (Life Technologies) and heatedat 80° C. for 20 minutes. After cooling for 15 minutes, Maxisorp 96-wellplates were coated with 100 ng/well of denatured PrP and incubated at2-8° C. overnight. After blocking with PBST/BSA, primary antibodies wereadded and incubated for 1 hour at room temperature. Remaining steps wereas described for anti-peptide ELISAs.

Maxisorp 96-well plates were coated overnight at 2-8° C. with 100ng/well of goat anti-His-6 antibody (QED) in PBS. After blocking withPBST/BSA, His-PrP (Alicon) was added and incubated for 1 hour at roomtemperature. Addition of primary antibody and remaining steps were asdescribed for anti-peptide ELISAs.

Adherent tumor cell lines and primary cells were detached from flasksusing non-enzymatic cell-dissociation buffer (Invitrogen). Peripheralblood mononuclear cells were prepared from fresh citrated blood on theday of collection using standard Ficoll centrifugation methods. Otherprimary cells were frozen in 10% DMSO and thawed on the day of testing.Implanted tumors were surgically removed from mice. Tumors were choppedwith scissors and then treated with collagenase/hyaluronidase(Worthington Biochemical) while shaking at 37° C. for 30 minutes.Individual tumor cells were collected by passing the mixture through a40 μm screen.

Cells with Fc receptors were treated with 10% normal human serum toblock the receptors. Cells were incubated with primary antibodies for 30minutes at 2-8° C. Following washing, cells were incubated with goatanti-rabbit AF488 for 30 minutes at 2-8° C. After the final wash, cellswere incubated in 1 μg/mL propidium iodide. Cells were analyzed usingeither a Becton Dickinson FACSCalibur or a Becton Dickinson FACS CantoII and FCS Express Software (DeNovo Systems).

The Octet QK system from ForteBio was used (by sub-contractor T-mab).Measurements were performed according to standard protocols.BSA-peptides were biotinylated and then coupled to a streptavidin-coatedbiosensor. The specific antibodies in the hybridoma supernatants wereallowed to incubate with the treated biosensor.

Binding of antibodies to denatured PrP was performed by ELISA asdescribed above. Binding of antibodies to tumor cells was performed byFACS as described above. Antibodies were titrated to provide bindingcurves. EC50 values were calculated using GraphPad software.

Results

The ProMis™ algorithm (described in WO 2010/040209) was used to identifyDSEs for human PrP. DSE3 is called the rigid loop epitope, and it islocated between β-sheet 2 and α-helix 1.

Anti-DSE3 antibodies were developed using the specific sequenceMDEYSNQNN, and two different immunogens, i.e., KLH-Cys-DSE3 andOVA-Cys-DSE3. Rabbits were immunized as described in the Materials andMethods and the antisera from the rabbits were evaluated. Rabbits madeexcellent responses to the immunogen peptides. In addition, antiserashowed excellent binding to full length denatured PrP. After performingfusions, monoclonal antibodies were generated. Seven recombinant rabbitmonoclonal antibodies raised against DSE3 were then fully evaluated.

Antibodies were tested for binding to the immunogen peptides, and allseven antibodies showed excellent titers. Kd values for peptide bindingwere determined using surface plasmon resonance. All antibodies showedvery high affinity for peptides, with Kds in the 10⁻¹⁰M range.

All antibodies were then tested by ELISA for binding to denaturedfull-length recombinant PrP. One antibody exhibited a titer fordenatured PrP that was similar to the anti-peptide affinities, in the10⁻¹⁰ M range. Thus, the preferred antibody exhibits preferably an EC50by this test that is at least better than 10⁻⁹M. The remainingantibodies showed logs lower affinity to denatured protein (10⁻⁶ to 10⁻⁹M range) than to peptide. All antibodies were also tested by ELISA forbinding to captured His-tagged PrP. None of the antibodies showedbinding to captured His-PrP.

All seven antibodies were tested for binding to a panel of eleven tumorcell lines, six implanted primary human tumors, and nine normal cells.Only two antibodies showed binding to tumor cell lines (DSE3 ab90 andDSE3 ab120). When tested for binding to normal cells, DSE3 ab90 showedmore binding to normal cells than to tumor cells. Although DSE3 ab120also showed a small amount of binding to normal cells, this was lessthan the amount of binding observed against both tumor cell lines andpassaged primary tumors. The binding of DSE ab120 was particularlystrong against ovarian tumor cells, as the antibody bound well to fiveof six ovarian tumors tested, but did not bind to normal ovarianepithelial cells. PrP is expressed on all ovarian cells tested, althoughto varying degrees. In order to account for the differences in overallPrP levels, the binding of DSE3 ab120 was normalized to the binding ofthe control PrP antibody, 6H4.

For the six tumor cells, normalized DSE3 ab120 binding ranged from a lowof 3.1 to a high of 71.6 (average=18.8), However, the normalized DSE3ab120 binding was only 0.4 for the normal ovarian cells.

In order to determine the affinity of DSE3 ab120 to tumor cells,antibody titrations were performed on three ovarian tumor cell lines.Antibody titrations were also performed on two types of normal cells,and confirmed the earlier findings that DSE3 ab120 does not bind tothese normal cells. Even though up to 40 ug/mL of antibody was tested,binding saturation was not reached on the tumor cells and affinitiescould not be determined. In the same experiments, the PrP controlantibody 6H4 was also titrated and binding saturation was reached. For6H4, the average calculated EC50 is 1.7×10⁻⁸M and there was nosignificant difference in the EC50 on tumor and normal cells. Since thebinding of DSE3 ab120 to tumor cells is of lower affinity than 6H4, theEC50 for DSE3 ab120 must be lower than 1.7×10⁻⁸ M, and thus at least onelog lower than the binding of DSE3 ab120 to denatured PrP (8.6×10⁻¹⁰ M).

The location of the CDRs within the antibody is determined by numberingamino acid residues with reference to the Kabat numbering system.

This antibody, herein designated ab120, thus displays an affinity forbinding to misfolded PrP when present on ovarian cancer cells. Itsability to detect senescent cells is explored in the next Example.

Example 5 Senescent Cell Detection, PrP-Based

When primary cells, HUVECs are induced to undergo senescence, there is aproportion of cell surface PrP protein that stains for amisfolding-specific epitope referenced herein as the rigid loop.

More particularly, HUVEC cells were seeded at 50% confluence in 6-wellculture plates and cultured in standard growth medium for 24 hours.Media was then removed and the cells were treated with 10 μg/mlMitomycin C for 2 hrs in fresh cell culture medium. The solution wasthen removed and the cells were washed twice in PBS for about 30 secondsper wash. Lysosomal alkalinization was induced using the SA-β-galstaining kit, according to the supplier's instructions (Cell Biolabs,CBA-232). The cells were ultimately washed three times in PBS. A cellscraper was used to harvest the cells, and blocking with PBS containing10% NGS for 30 minutes on ice. Those cells were then stained by firstincubating the cells on ice with primary antibodies, i.e., antibodyc-120 at 40 μg/ml and antibody 6D11 as control at 5 μg/ml, washing incold PBS/2% NGS, and then incubating for 30 minutes on ice withsecondary Alexa-488 labeled antibodies. Cells were then subjected toflow cytometry and analyzed. Results are shown in FIGS. 1 and 2.

When treated and untreated cells were incubated with either (1) astandard murine antibody (mIgG), (2) a PrP antibody that binds PrP inboth natively and misfolded conformations (6D11), or (3) the c-120 PrPantibody that binds the rigid loop of PrP and thus binds selectively toPrP in a misfolded state, it was revealed (FIG. 2) that senescent HUVECswere bound selectively by the misfolded PrP antibody.

Example 6 Senescent Cell Detection, FasR-Based

The principles established herein are further demonstrated by targetinganother and different cell surface protein first to determine if it,too, adopts a misfolded conformation when present on senescent cells,and then to determine if antibodies to that target can inhibit thegrowth of those target-positive cells.

Chosen as a second target was the protein FasR. Fas receptor (FasR) isknown also as human tumour necrosis factor superfamily member 6 receptor(hTNFRSM6), and as CD95, and is implicated in cancer. It is a deathreceptor on the surface of of cells that leads to caspase-mediatedprogrammed cell death (apoptosis). Antibodies to a misfolded form ofthis protein were prepared based on the epitopes predicted by Cashman etal, WO 2010/040209, as shown in Table 3:

TABLE 3 Human FASR (hTNFR6) 52-60 LHHDGQFCH 62 P25445 residues 1- 70-80ARDCTVNGDEP 63 335 105-111 RLCDEGH 64 136-142 NSTVCEH 65 167-189EEPSRSNLGWLCL 66

To raise antibodies against misfolded FasR, peptides comprising the FasRepitope sequences LHHDGQFCH (SEQ ID No. 62) and NSTVCEH (SEQ ID No. 65)were synthesized using standard methods and then coupled to carrierproteins. Prepared immunogens included both KLH-Cys-X and OVA-Cys-X,where X is NSTVCEH or LHHDGQFCH.

New Zealand white rabbits were immunized subcutaneously with 0.4 mgpeptide-KLH conjugates in complete Freund's adjuvant. After the initialimmunization, animals were boosted several times every 2-3 weeks. Therabbit with the best titer in immunoassay was intravenously boosted withpeptide antigen again, four days before the removal of the spleen. Thehybridoma fusion was performed using conventional PEG cell fusionmethodology. Splenocytes were harvested from the immunized rabbit andfused with rabbit plasmacytoma cells 240E-W2 (U.S. Pat. No. 5,675,063)using PEG4000 (Sigma Chemical, St. Louis, Mo.) and selected by HAT(hypoxanthine, aminopterin, and thymidine). At the end of selectionhybridoma supernatants were collected and evaluated in various assays.Selected hybridomas were subsequently subcloned by limited dilution toobtain monoclonal hybridomas.

The antibody heavy and light chain genes for monoclonal AMF-3a-118 andAMF-3d-19 were cloned from the hybridoma cells. Total RNA was extractedand reverse-transcribed to cDNA using the Qiagen TurboCapture mRNA kits.DNA fragments for L chain and the variable region (VH) of H chain ofrabbit IgG were amplified by PCR with rabbit H and L chain primers. TheL chain fragment was cloned into pTT5 mammalian expression vector andthe VH fragment fused in-frame to the constant region of H chain pTT5Heavy chain vector For each hybridoma clone, three plasmid DNA clonesfor H and L chains were sequenced and expressed as recombinant RabMAbfor characterization.

Plamids encoding the IgG heavy and light chains of AMF-3a-118 andAMF-3d-19 were isolated from transformed E. coli using EndoFree® plasmidpurification kit (Qiagen). Human HEK-293-6E cells were used fortransient expression of AMF-3a-118 and AMF-3d-19 antibodies. Theantibody plasmids were transfected into cells at logarithmic growthphase using FreeStyle™ MAX Reagent 293 fectin (Invitrogen, Cat: 51-0031)and cultured in FreeStyle™ 293 Expression Medium (Invitrogen, Cat:12338-18) according to manufacturer's instructions. The antibodysecreted into the culture medium was collected by spinning at 7000 rpmfor 15 minutes to remove cell debris. The cleared culture supernatantwas purified by protein A chromatography (HiTrap™ rProtein A FF, GEhealthcare, CAT: 17-5080-01) under endotoxin free condition. Antibodieswere eluted from the column in citrate elution buffer (SIGMA, CAT:C2404-100G) and adjusted to neutral pH with sodium bicarbonate buffer.The antibody preparations were concentrated and exchanged into PBSbuffer.

Purified antibody was filter-sterilized and stored at 4° C. in PBSbuffer (pH 7.4). The protein concentration was determined by UVabsorption 280 nm) assay and PBS buffer was used a blank buffer. Tomeasure protein purity, SDS-PAGE was performed with Bio-Rad minielectrophoresis system according to the manufacturer's instructions. Thegel was then stained with Coomassie brilliant blue. The resolving gelwas 12% acrylamide and the stacking gel was 4% acrylamide. The assayedsamples showed 2 bands (Heavy chain and Light chain) in reducedSDS-PAGE, and one band (whole IgG molecule) in non-reduced SDS-PAGE.

Thus in a preferred embodiment, the antibody is provided as apreparation that exhibits (a) a concentration of greater than about 1mg/ml, (b) and migration as a single protein band when measured bynon-reducing SDS-PAGE.

Maxisorp 96-well plates were coated overnight at 2-8° C. with 100ng/well of BSA-peptide in PBS. After blocking with PBST/casein, primaryantibodies were added and incubated for 1 hour at room temperature.Rabbit antibodies were detected using goat anti-rabbit IgG-HRP and TMBsubstrate. After stopping the reaction with 0.25M sulfuric acid,absorbance was measured at 450 nm.

Recombinant Fas extracellular domain-Fc fusion protein (Aragen) wasmixed with LDS sample buffer (Life Technologies) and sample reducingagent (Life Technologies) and heated at 80° C. for 20 minutes. Aftercooling for 15 minutes, Maxisorp 96-well plates were coated with 100ng/well of denatured Fas and incubated at 2-8° C. overnight. Afterblocking with PBST/BSA, primary antibodies were added and incubated for1 hour at room temperature. Remaining steps were as described foranti-peptide ELISAs.

His-tagged Fas (Creative Biomart) was mixed with Talon Dynabeads(Invitrogen) in round-bottom polystyrene 96-well plates for 30 minutesat room temperature. After beads were collected on a magnet, beads werewashed 3× with PBST, then mixed with primary antibodies at roomtemperature for 60 minutes. Bound primary antibodies were detected withgoat anti-rabbit IgG-HRP and TMB substrate. Supernatant was transferredto a flat-bottom 96-well plate (Nunc), the reaction stopped with 0.25Msulfuric acid, and absorbance was measured at 450 nm.

Antibodies having particularly good binding profiles include antibodyAMF-3a-118 for the peptide LHHDGQFCH, and antibody AMF-3d-19 for thepeptide NSTVCEH. These antibodies have the full length protein sequencesset out in SEQ ID No. 91 (light chain) and SEQ ID No.89 (heavy chain)for 3a-118, and sequences set out in SEQ ID No. 105 (light chain) andSEQ ID No.103 (heavy chain) for 3d-19. Polynucleotides encoding themhave the sequences set out in SEQ ID No. 99 (heavy chain) and SEQ IDNo.101 (light chain) for 3a-118 and SEQ ID No. 113 (heavy chain) and SEQID No. 115 (light chain) for antibody 3d-19. The importantcomplementarity determining regions (CDRs) of these antibodies are setout below:

AMF-3a-118

For the heavy chain:

CDR1 (SEQ ID No. 93) CDR2 (SEQ ID No. 94) CDR3 (SEQ ID No. 95)For the light chain:

CDR1 (SEQ ID No. 96) CDR2 (SEQ ID No. 97) CDR3 (SEQ ID No. 98)

AMF-3d-19

For the heavy chain:

CDR1 (SEQ ID No. 107) CDR2 (SEQ ID No. 108) CDR3 (SEQ ID No. 109)For the light chain:

CDR1 (SEQ ID No. 110) CDR2 (SEQ ID No. 111) CDR3 (SEQ ID No. 112)

These misfolded FasR antibodies were assessed for their ability todetect senescent cells, using the approach already described above. Moreparticularly, HUVEC cells were seeded at 50% confluence in 6-wellculture plates and cultured in standard growth medium for 24 hours.Media was then removed and the cells were treated with 10 μg/mlMitomycin C for 2 hrs in fresh cell culture medium. The solution wasthen removed and the cells were washed twice in PBS for about 30 secondsper wash. Lysosomal alkalinization was induced using the SA-β-galstaining kit, according to the supplier's instructions (Cell Biolabs,CBA-232). The cells were ultimately washed three times in PBS. A cellscraper was used to harvest the cells, and blocking with PBS containing10% NGS for 30 minutes on ice. Those cells were then stained by firstincubating the cells on ice with primary antibodies, i.e., antibodies3a-118 and 3d-19 at 40 μg/ml and antibody 6D11 as control at 5 μg/ml,washing in cold PBS/2% NGS, and then incubating for 30 minutes on icewith secondary Alexa-488 labeled antibodies. Cells were then subjectedto flow cytometry and analyzed. Results are shown in FIGS. 3 and 4.

When treated and untreated cells were incubated with either (1) astandard murine antibody (mIgG), (2) a PrP antibody that binds PrP inboth natively and misfolded conformations (6D11), or (3) the FasRantibodies that binds the noted exposed epitopes and thus bindselectively to FasR in a misfolded state, it was revealed (FIGS. 3 and4) that senescent HUVECs were bound selectively by the misfolded FasRantibodies.

All citations are herein incorporated by reference, as if eachindividual publication was specifically and individually indicated to beincorporated by reference herein and as though it were fully set forthherein.

Table of Sequences SEQ ID Subject Sequence 1 Misfolded PrP epitopeLGGYML 2 Misfolded PrP epitope GGYMLGS 3 Misfolded PrP epitope HFGSDYED4 Misfolded PrP epitope SDYED 5 Misfolded PrP epitope RYYRENMH 6Misfolded PrP epitope RENMH 7 Misfolded PrP epitope QVYYRPM 8Misfolded PrP epitope PMDEYSNQNN 9 Misfolded PrP epitope MDEYSNQNN 10Misfolded PrP epitope KQHTVTTTTKGEN 11 Misfolded PrP epitope ARDCTVNGDEP12 Misfolded PrP epitope RLCDEGH 13 Misfolded PrP epitope NSTVCEH 14Misfolded PrP epitope EEPSRSNLGWLCL 15 Misfolded PrP sequence RYYRE 16Misfolded PrP sequence RYYREN 17 Misfolded PrP sequence RYYRENM 18Misfolded PrP sequence DRYYRENMH 19 Misfolded PrP sequence DRYYRENM 20Misfolded PrP sequence VYYRPM 21 Misfolded PrP sequence QVYYRP 22Misfolded PrP sequence QVYYR 23 Misfolded PrP sequence QVYYRPMD 24Misfolded PrP sequence QVYYRPM 25 Misfolded PrP sequence GGYMLG 26Misfolded PrP sequence GYMLGS 27 Misfolded PrP sequence GGYML 28Misfolded PrP sequence YMLGS 29 Misfolded PrP sequence GYML 30Misfolded PrP sequence YMLG 31 Misfolded PrP sequence LGGYML 32Misfolded PrP sequence LGGYMLG 33 Misfolded PrP sequence YML 34Misfolded PrP sequence PMDEYSNQNN 35 Misfolded PrP sequence DEYSNQNN 36Misfolded PrP sequence MDEYSNQ 37 Ab c-120 light chainMDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQSSQSLYNKNWLSWYQKKPGQPPKLLIYKASTLESGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSADCTAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC 38 Ab c-120 heavy chainMETGLRWLLLVAVLKGVQCQSVEESGGHLVTPGTPLTLTCTVSGIDLSTYAMGWVRQAPGKGLEWIGVITKSGNTYYASWAKGRFAISKTSTTVDLKITSPTTEDTATYFCGRYGIGVSYYDIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK 39Ab c-120 light chain variable region (VL)MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQSSQSLYNKNWLSWYQKKPGQPPKLLIYKASTLESGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSADCTAFGGGTEVVV 40Ab c-120 heavy chain variable region (VH)METGLRWLLLVAVLKGVQCQSVEESGGHLVTPGTPLTLTCTVSGIDLSTYAMGWVRQAPGKGLEWIGVITKSGNTYYASWAKGRFAISKTSTTVDLKITSPTTEDTATYFCGRYGIGVSYYDIWGPGTLVTVSSGQ 41Ab c-120 CDR1 heavy TYAMG 42 Ab c-120 CDR2 heavy VITKSGNTYYASWAKG 43Ab c-120 CDR3 heavy YGIGVSYYDI 44 Ab c-120 CDR1 light QSSQSLYNKNWLS 45Ab c-120 CDR2 light KASTLES 46 Ab c-120 CDR3 light QGEFSCSSADCTA 47misfolded CD44 epitope NGRYSIS 48 misfolded CD44 epitope EGHVV 49misfolded CD44 epitope TSNTSNYDT 50 misfolded CD44 epitope NRDGTRYVQKG51 misfolded CD44 epitope ANNTFVYILTSNTSNYDT 52 misfolded hTNFR epitopeIHPQNNSICCT 53 misfolded hTNFR epitope NDCPGPGQDTD 54misfoided hTNFR epitope YWSENLF 55 misfoided hTNFR epitope HLSCQEKQN 56misfolded hTNFR epitope CTCHAGFFLRENECV 57 misfolded hNOTCH1 epitopeCEHAGKC 58 misfolded hNOTCH1 epitope MPGYEGVHC 59misfolded hNOTCH1 epitope CLHNGRC 60 misfolded hNOTCH1 epitope KINEF 61misfolded hNOTCH1 epitope ECASSPCLHNGRCLDKINEFQCECP 62misfolded hFas epitope LHHDGQFCH 63 misfolded hFas epitope ARDCTVNGDEP64 misfolded hFas epitope RLCDEGH 65 misfolded hFas epitope NSTVCEH 66misfolded hFas epitope EEPSRSNLGWLCL 67 misfolded hEGFR epitopeSNKLTQLFTFEDHFL 68 misfolded hEGFR epitope VQRNYDLSFLK 69misfolded hEGFR epitope IRGNMYYENSYAL 70 misfolded hEGFR epitopeVLSNYDANKTG 71 misfolded hEGFR epitope IVSSDFLSNMSMD 72misfolded hEGFR epitope FLSNMSMDFQNHLGS 73 misfolded hEGFR epitopeWGAGEE 74 misfolded hEGFR epitope PPLMLYNPTTYQMDVNPE 75misfolded hEGFR epitope PEGKYSFGAT 76 misfolded hEGFR epitope RNYVVTDHGS77 misfolded hEGFR epitope GADSYEMEEDGVRK 78 misfolded hEGFR epitopeNGIGIGEFKDSLSIN 79 misfolded hEGFR epitope ATNIKHFKN 80misfolded hEGFR epitope LPVAFRGDSFTHTPPL 81 misfolded hEGFR epitopeKIISNRGENS 82 misfolded CD38 epitope YTEIHPEMRHVDCQS 83misfolded CD38 epitope GEFATSKIN 84 misfolded CD38 epitope WRKDCSN 85misfolded CD38 epitope GSRSKIFDKDS 86 misfolded CD38 epitope IHGGREDSRDL87 DNA for ab c-120 heavy chainATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCACCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTACCTATGCAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTACTAAAAGTGGTAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCGCCATCTCCAAAACCTCGACCACGGTGGATCTAAAGATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGATATGGTATTGGTGTTTCTTACTATGACATCTGGGGCCCAGGCACTCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCGAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 88DNA coding for c-120 light chainATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCATCCCCTGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTCTTTATAATAAGAACTGGTTATCCTGGTATCAGAAGAAACCAGGGCAGCCTCCTAAGCTCCTGATCTACAAGGCATCCACTCTGGAATCTGCGGTCTCATCGCGGTTCAAGGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCGAATTTAGTTGTAGTAGTGCTGATTGTACGGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG 89 AMF-3a-118 heavy chainM E T G L R W L L L V A V L K G V Q C QS V E E S G G R L V T P G T P L T L T CK A S G F S L S D S R V S W V R Q A P GK G L E W I G I V G I G W N I Y H A N WA K G R F T I S K T S S T T V D L K I TS P T V E D T A T Y F C A R G L G G G TV I W G P G T L V T V S L G Q P K A P SV F P L A P C C G D T P S S T V T L G CL V K G Y L P E P V T V T W N S G T L TN G V R T F P S V R Q S S G L Y S L S SV V S V T S S S Q P V T C N V A H P A TN T K V D K T V A P S T C S K P T C P PP E L L G G P S V F I F P P K P K D T LM I S R T P E V T C V V V D V S Q D D PE V Q F T W Y I N N E Q V R T A R P P LR E Q Q F N S T I R V V S T L P I A H QD W L R G K E F K C K V H N K A L P A PI E K T I S K A R G Q P L E P K V Y T MG P P R E E L S S R S V S L T C M I N GF Y P S D I S V E W E K N G K A E D N YK T T P A V L D S D G S Y F L Y S K L SV P T S E W Q R G D V F T C S V M H E AL H N H Y T Q K S I S R S P G K — 90AMF-3a-118 heavy chain variable regionM E T G L R W L L L V A V L K G V Q C QS V E E S G G R L V T P G T P L T L T CK A S G E S L S D S R V S W V R Q A P GK G L E W I G I V G I G W N I Y H A N WA K G R F T I S K T S S T T V D L K I TS P T V E D T A T Y F C A R G L G G G T V I W G P G T L V T V S L 91AMF-3a-118 light chain M D T R A P T Q L L G L L L L W L P G AT F A Q V L T Q T P A S V S A A V G G TV T I S C Q S S E S V Y K N N Y L S W FQ Q K P G Q P P K L L I Y E A S K L A SG V S T R F K G S G S G T Q F T L T I SG V Q C D D A A T Y Y C L G E F S C Y SG D C G T F G G G T A V V V K G D P V AP T V L I F P P A A D Q V A T G T V T IV C V A N K Y F P D V T V T W E V D G TT Q T T G I E N S K T P Q N S A D C T YN L S S T L T L T S T Q Y N S H K E Y TC K V T Q G T T S V V Q S F N R G D C — 92AMF-3a-118 light chain variable regionM D T R A P T Q L L G L L L L W L P G AT F A Q V L T Q T P A S V S A A V G G TV T I S C Q S S E S V Y K N N Y L S W FQ Q K P G Q P P K L L I Y E A S K L A SG V S T R F K G S G S G T Q F T L T I SG V Q C D D A A T Y Y C L G E F S C Y S G D C G T F G G G T A V V V K 93Ab 3a-118 CDR1 heavy DSRVS 94 Ab 3a-118 CDR2 heavy IVGIGWNIYHANWAKG 95Ab 3a-118 CDR3 heavy GLGGGTVI 96 Ab 3a-118 CDR1 light QSSESVKNNYLS 97Ab 3a-118 CDR2 light EASKLAS 98 Ab 3a-118 CDR3 light LGEFSCYSGDCGT 99AMF-3a-118 heavy chain-encoding DNAATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTGACTCTAGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCGTTGGCATTGGTTGGAATATATACCACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACGTCGTCGACCACGGTGGATTTGAAAATCACCAGTCCGACAGTCGAGGACACGGCCACCTATTTCTGTGCCAGAGGTCTGGGTGGTGGTACTGTCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTTAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 100AMF-3a-118 heavy chain variable region-encoding DNAATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTGACTCTAGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCGTTGGCATTGGTTGGAATATATACCACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACGTCGTCGACCACGGTGGATTTGAAAATCACCAGTCCGACAGTCGAGGACACGGCCACCTATTTCTGTGCCAGAGGTCTGGGTGGTGGTACTGTCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTTA 101 AMF 3a-118 light chain-encoding DNAATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTGAGAGTGTTTATAAGAACAACTACTTATCCTGGTTTCAGCAGAAACCAGGACAGCCTCCCAAGCTCCTGATCTACGAAGCATCCAAACTGGCATCTGGGGTCTCAACGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACATACTACTGTCTAGGCGAATTTAGTTGTTATAGTGGTGATTGTGGTACTTTCGGCGGAGGGACCGCGGTGGTGGTCAAAGCTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTCATCAGGTGGCAACTGCAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG 102 AMF 3a-118 light chain variable region-encoding DNAATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTGAGAGTGTTTATAAGAACAACTACTTATCCTGGTTTCAGCAGAAACCAGGACAGCCTCCCAAGCTCCTGATCTACGAAGCATCCAAACTGGCATCTGGGGTCTCAACGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACATACTACTGTCTAGGCGAATTTAGTTGTTATAGTGGTGATTGTGGTACTTTCGGCGGAGGGACCGCGGTGGTGGTCAAA 103 AMF3d-19 heavy chainM E T G L R W L L L V A V L K G V Q C QS L E E S G G R L V T P G T P L T L T CT V S G F S L S R N A I N W V R Q A P GK G L E Y I G I I G S S G V T Y Y A S WA K G R F T I S R T S T T V D L K I T SP T T E D T A T Y F C A R N L Y T G G SN D N L W G P G T L V T V S S G Q P K AP S V F P L A P C C G D T P S S T V T LG C L V K G Y L P E P V T V T W N S G TL T N G V R T F P S V R Q S S G L Y S LS S V V S V T S S S Q P V T C N V A H PA T N T K V D K T V A P S T C S K P T CP P P E L L G G P S V F I F P P K P K DT L M I S R T P E V T C V V V D V S Q DD P E V Q F T W Y I N N E Q V R T A R PP L R E Q Q F N S T I R V V S T L P I AH Q D W L R G K E F K C K V H N K A L PA P I E K T I S K A R G Q P L E P K V YT M G P P R E E L S S R S V S L T C M IN G F Y P S D I S V E W E K N G K A E DN Y K T T P A V L D S D G S Y F L Y S KL S V P T S E W Q R G D V F T C S V M HE A L H N H Y T Q K S I S R S P G K — 104AMF3d-19 heavy chain variable regionM E T G L R W L L L V A V L K G V Q C QS L E E S G G R L V T P G T P L T L T CT V S G F S L S R N A I N W V R Q A P GK G L E Y I G I I G S S G V T Y Y A S WA K G R F T I S R T S T T V D L K I T SP T T E D T A T Y F C A R N L Y T G G S N D N L W G P G T L V T V S S105 AMF 3d-19 light chain M D T R V P T Q L L G L L L L W L P G AT F A Q V L T Q T P S P V S A A V G G TV T I N C Q A S K S V y N N V Q L S W FQ Q K P G Q P P K R L I Y Y A S T L A SG V P S R F K G S G S G T Q F T L T I SD V Q C D D V A T Y Y C A G G Y S S S SD N A F G G G T E V V V K G D P V A P TV L I F P P A A D Q V A T G T V T I V CV A N K Y F P D V T V T W E V D G T T QT T G I E N S K T P Q N S A D C T Y N LS S T L T L T S T Q Y N S H K E y T C KV T Q G T T S V V Q S F N R G D C — 106AMF 3d-19 light chain variable regionM D T R V P T Q L L G L L L L W L P G AT F A Q V L T Q T P S P V S A A V G G TV T I N C Q A S K S V Y N N V Q L S W FQ Q K P G Q P P K R L I Y Y A S T L A SG V P S R F K G S G S G T Q F T L T I SD V Q C D D V A T Y Y C A G G Y S S S S D N A F G G G T E V V V K 107Ab 3d-19 CDR1 heavy RNAIN 108 Ab 3d-19 CDR2 heavy ITGSSGVTYYASWAKG 109Ab 3d-19 CDR3 heavy NLYTGGSNDNL 110 Ab 3d-19 CDR1 light QASKSVYNNVQLS111 Ab 3d-19 CDR2 light YASTLAS 112 Ab 3d-19 CDR3 light AGGYSSSSDNA 113AMF 3d-19 heavy chain-encoding DNAATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGTTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAGTCGCAATGCAATAAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTGGTAGTAGTGGTGTCACATACTACGCGAGCTGGGCAAAAGGCCGATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTTTGTGCCAGAAATCTTTATACTGGTGGTAGTAATGATAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 114AMF 3d-19 heavy chain variable region-encoding DNAATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGTTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAGTCGCAATGCAATAAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTGGTAGTAGTGGTGTCACATACTACGCCAGCTGGGCAAAAGCCCGATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTTTGTGCCAGAAATCTTTATACTGGTGGTAGTAATGATAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA 115 AMF 3d-19 light chain-encoding DNAATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTGACTCTAGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCGTTGGCATTGGTTGGAATATATACCACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACGTCGTCGACCACGGTGGATTTGAAAATCACCAGTCCGACAGTCGAGGACACGGCCACCTATTTCTGTGCCAGAGGTCTGGGTGGTGGTACTGTCATCTGGGGCCCAGGCACCCTGCTCACCGTCTCCTTAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 116AMF 3d-19 light chain variable region-encoding DNAATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGCTGTCCAGTGTCAGTCGGTGGAGGACTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTGACTCTAGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAACCGGCTGGAATGGATCGGAATCGTTGGCATTGGTTGGAATATATACCACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACGTCGTCGACCACGGTGGATTTGAAAATCACCAGTCCGACAGTCGAGGACACGGCCACCTATTTCTGTGCCAGAGGTCTGGGTGGTGGTACTGTCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTTA 119 DNA for ab c-120 heavy chain (lower case =non-coding restriction site) aagcttgtacccttcaccATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCACCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTACCTATGCAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTACTAAAAGTGGTAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCGCCATCTCCAAAACCTCGACCACCGTGGATCTAAAGATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGATATGGTATTGGTGTTTCTTACTATGACATCTGGGGCCCAGGCACTCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTCCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGAgcgctgtgccggcgagctgcggccgc 120DNA coding for c-120 light chain (lower case = non-coding restrictionsite) aagcttgtacccttcaccATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCATCCCCTGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTCTTTATAATAAGAACTGGTTATCCTGGTATCAGAAGAAACCAGGGCAGCCTCCTAAGCTCCTGATCTACAAGGCATCCACTCTGGAATCTGGGGTCTCATCGCGGTTCAAGGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCGAATTTAGTTGTAGTAGTGCTGATTGTACGGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG agtgagagcggccgc 121AMF-3a-118 heavy chain-encoding DNA (sequence before ATG and after TGA,vector sequence; underlined sequence restriction site) AAGCTTGTACCCTTCACCATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTGACTCTAGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCGTTGGCATTGGTTGGAATATATACCACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACGTCGTCGACCACGGTGGATTTGAAAATCACCAGTCCGACAGTCGAGGACACGGCCACCTATTTCTGTGCCAGAGGTCTGGGTGGTGGTACTGTCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTTAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGGAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA GCGCTGTGCCGGCGAGCTGCGGCCGC 122AMF 3a-118 light chain-encoding DNA (sequence before ATG and after TAG,vector sequence; underlined sequence restriction site)aagcttgtacccttcaccATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTGAGAGTGTTTATAAGAACAACTACTTATCCTGGTTTCAGCAGAAACCAGGACAGCCTCCCAAGCTCCTGATCTACGAAGCATCCAAACTGGCATCTGGGGTCTCAACGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACATACTACTGTCTAGGCGAATTTAGTTGTTATAGTGGTGATTGTGGTACTTTCGGCGGAGGGACCGCGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG Agtgaga gcggccgc 123AMF 3d-19 heavy chain-encoding DNA (sequence before ATG and after TGA,vector sequence; underlined sequence restriction site) aagcttgtacccttcaccATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGTTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAGTCGCAATGCAATAAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTGGTAGTAGTGGTGTCACATACTACGCGAGCTGGGCAAAAGGCCGATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTTTGTGCCAGAAATCTTTATACTGGTGGTAGTAATGATAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTCGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTCGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGCGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAACGACACCCTCATGATCTCACCCACCCCCGAGCTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCACTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGCGAGAAGAACCGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA Gcgctgtgccggcgagctgcggccgc 124AMF 3d-19 light chain-encoding DNA (sequence before ATG and after TGA,vector sequence; underlined sequence restriction site) aagcttgtacccttcaccATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCCCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTGACTCTAGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATCGATCCGAATCGTTGGCATTGGTTGGAATATATACCACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACGTCGTCGACCACGGTGGATTTGAAAATCACCAGTCCGACAGTCGAGGACACGGCCACCTATTTCTGTGCCAGACGTCTGGGTGGTGCTACTGTCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTTAGGGCAACCTAACGCTCCATCAGTCTTCCCACTGCCCCCCTGCTGCGGCGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCCCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA gcgctgtgccggcgagctgcggccgc

1. A method for depleting senescent cells endogenous to a subject,comprising administering to the subject a binding agent that isselectively toxic to senescent cells in an amount effective to reducethe number of such cells, wherein the binding agent binds selectively toa senescent cell surface protein having a misfolded conformation,relative to said protein in a native conformation.
 2. The methodaccording to claim 1, wherein the binding agent binds selectively to amisfolded form of human PrP.
 3. The method according to claim 2, whereinthe binding agent binds a misfolded human PrP epitope comprising a YYRmotif or a YML motif.
 4. (canceled)
 5. The method according to claim 2,wherein the binding agent binds a misfolded human PrP epitope comprisingMDEYSNQNN (SEQ ID No.9).
 6. The method according to claim 1, wherein thebinding agent binds selectively to a misfolded form of human Fasreceptor.
 7. The method according to claim 6, wherein the binding agentbinds a misfolded human Fas receptor epitope comprising LHHDGQFCH (SEQID No. 62) or NSTVCEH (SEQ ID No. 65).
 8. (canceled)
 9. The methodaccording to claim 1, wherein the binding agent binds selectively to amisfolded form of a surface protein selected from the human forms of Fasligand, CD44, EGF receptor, CD38, Notch-1, CD44, CD59, and TNF receptor.10. The method according to claim 1, wherein the binding agent is anantibody or a binding fragment thereof.
 11. The method according toclaim 10, wherein the antibody is a misfolded human PrP antibody thatbinds selectively to an epitope having the sequence of any one of SEQ IDNos. 1-14.
 12. The method according to claim 11, wherein the misfoldedhuman PrP antibody is an antibody produced by, or that binds to the sameepitope as the antibody produced by the hybridoma cell line depositedwith the International Depositary Authority of Canada under accessionnumber 260210-01.
 13. The method according to claim 11, wherein themisfolded human PrP antibody is an antibody comprising the followingCDRs: For the heavy chain: (SEQ ID No. 41) CDR1 TYAMG (SEQ ID No. 42)CDR2 VITKSGNTYYASWAKG (SEQ ID No. 43) CDR3 YGIGVSYYDI

For the light chain: (SEQ ID No. 44) CDR1 QSSQSLYNKNWLS (SEQ ID No. 45)CDR2 KASTLES (SEQ ID No. 46) CDR3 QGEFSCSSADCTA.


14. The method according to claim 13, wherein the antibody is theantibody c-120.
 15. The method according to claim 10, wherein theantibody is a misfolded human Fas receptor antibody that bindsselectively to an epitope having the sequence of any one of SEQ ID Nos.62-66.
 16. The method according to claim 1, for the treatment of anage-related degenerative disorder.
 17. The method according to claim 16,for the treatment of cancer, or emphysema, or aging of the skin, orsarcopenia. 18.-20. (canceled)
 21. The method according to claim 1,wherein the binding agent is a conjugate comprising an antibody orbinding fragment thereof, and a second agent that is a toxin or adetectable label.
 22. (canceled)
 23. A method for detecting senescentcells, comprising combining (1) a binding agent that binds selectivelyto a senescent cell surface protein having a misfolded conformation, and(2) a sample containing cells to be screened, and then determining theformation of a complex therebetween, the presence of such complexindicating that the sample contains a senescent cell.
 24. The methodaccording to claim 24, wherein the binding agent further comprises adetectable label.
 25. The method according to claim 23, wherein thesenescent state of the cell is confirmed by a second senescent celldetection method; optionally wherein the second senescent cell detectionmethod is a test for B-galactosidase activity at pH6.
 26. (canceled) 27.A method for imaging senescent cells in a subject, comprisingadministering to the subject an imaging agent comprising a detectablelabel and, conjugated thereto, a binding agent that binds selectively toa senescent cell surface protein having a misfolded conformation,allowing the imaging agent to bind to any such senescent cells, anddetecting the location of the detectable label in said subject.
 28. Adiagnostic kit comprising (1) an antibody that binds selectively to asenescent cell surface protein having a misfolded conformation, and (2)written instructions for the use thereof to detect senescent cells inaccordance with the method defined in claim 23.